Patent Application: US-43222303-A

Abstract:
the present invention relates to the demonstration of a direct relationship between the amount of trna lys3 packaged into hiv , the amount of trna lys3 placed onto the reverse transcriptase primer binding site which can initiate reverse transcription , and viral infectivity . the present invention also relates to the incorporation of lysyl trna synthase into hiv - 1 and to the aminoacylation status of trna lys3 and its impact on virion incorporation . the present invention also relates to methods of modulating lysyl trna synthetase - facilitated processes associated with trna lys3 priming function of rt , to bioassays to screen and identify compounds which interfere with these processes and to compositions for modulating these processes . in one particular embodiment , the compositions modulate the incorporation of lysrs and / or trna lys3 into hiv and related virions . the present invention also relates to aars - facilitated processes associated with their cognate trna aa priming function in other types of retroviruses and to methods , assays and compositions which modulate them .

Description:
in view of the different protein - protein and protein - rna interactions involved in ensuring that proper functioning of aminoacyl trna synthetase - processes associated with its cognate trna priming function occur , modulation of these processes can be effected in a number of ways , keeping in mind that 1 ) aminoacyl trna synthetase is the signal for its cognate trna packaging , 2 ) that the level of incorporation of trna packaging correlates with its aminoacylation ; and 3 ) that cleavage of the synthetase which occurs in the virion may a ) free the trna for annealing to the viral genomic rna and b ) cause deacylation of aminoacylated trna so that it can act as a primer for reverse transcription . herein , the relationship between viral trna lys3 concentration and its placement onto the primer binding site ( pbs ) was analyzed by making use of naturally occurring variation in viral trna lys3 concentration that we find in different virus preparations . the combination of trna lys3 was artificially increased and decreased by the cytoplasmic synthesis of excess trna lys3 or trna lys2 , using cells transfected with plasmids coding for these trnas as well as for hiv - 1 . in virus from both transfected cos cells and infected cell lines , a direct correlation was found between viral trna lys3 concentration , trna lys3 - primed initiation of reverse transcription , and infectivity of the viral population . during hiv - 1 assembly , both trna lys and lysyl trna synthetase ( lysrs ) are incorporated into hiv - 1 . the lysrs is resistant to digestion with the protease subtilisin , and searches for two other amino acyl trna sythetases , prors and ilers , revealed their absence in the virion . while the major cytoplasmic species of lysrs in infected cells has an mr = 70000 kd ( large species ), viral incorporation of trna lys is correlated with the packaging of an intermediate size lysrs species , mr = 63000 kd . this intermediate species is the major form of lysrs found in virions produced from chronically infected cells ( h9 , u937 , plb , cemss ), while in wild type or protease - negative hiv - 1 produced from cos cells ( hiv ( cos )), both the large and intermediate lysrs species are found . the presence of the intermediate size lysrs in protease - negative viruses indicates that a cellular protease is involved . the intermediate lysrs species becomes the major lysrs species in hiv ( cos ) when viral trna lys3 packaging is increased as a result of a cotransfection of cos cells with hiv - 1 proviral dna and a trna lys3 gene . in mutant hiv ( cos ) which are defective in trna lys3 packaging ( p31l ( nc mutation ), dr2 ( rt mutation ), and gag - only particles ), no intermediate size lysrs species is detected . rescue of trna lys3 packaging in the p31l mutant with wild type gag - pol also results in an increase in the incorporation of the intermediate form of lysrs within the virus . trna lys packaging in hiv is shown herein to be limited by lysrs , since the overproduction of lysrs from a cotransfected plasmid encoding lysrs results in up to a 2 fold increase in a ) the incorporation of both trna lys isoacceptors into the viruses , b ) increased placement on the viral genome , and c ) increased viral infectivity . overproduction of a mutant lysrs lacking the n terminal 65 amino acids also results in increases in lysrs viral packaging , but no increase in trna lys viral packaging is observed , since the mutant lysrs cannot bind to trnalys . the present invention is illustrated in further detail by the following non - limiting examples . correlation between the viral trna concentration in the hiv virion , the level of initiation of reverse transcriptase and hiv infectivity during retroviral assembly , particular species of cellular trna are selectively packaged into the virus , where they are placed onto the primer binding site ( pbs ) of the viral genome , and are used to initiate the reverse - transcriptase - catalyzed synthesis of minus strand cdna . trna trp is the primer for all members of the avian sarcoma and leukosis virus group examined to date ( faras et al ., 1975 ; harada et al ., 1975 ; peters et al ., 1980 ; sawyer et al ., 1973 ; waters et al ., 1977 ; waters et al ., 1975 ). the common primer trnas in mammalian retroviruses are trna pro and trna lys . trna pro is the common primer for murine leukemia virus ( mulv ) ( harada et al ., 1979 ; peters et al ., 1977 ; taylor et al ., 1977 ). in mammalian cells , there are three major trna lys isoacceptors ( raba et al ., 1979 ). trna lys1 , 2 , representing two trna lys isoacceptors differing by one base pair in the anticodon stem , is the primer trna for several retroviruses , including mason - pfizer monkey virus ( mpmv ) and human foamy virus ( hfv ) ( leis et al ., 1993 ). trna lys3 serves as the primer for mouse mammary tumor virus ( peters et al ., 1980 ; waters et al ., 1978 ), and the lentiviruses such as equine infectious anemia virus ( eiav ), feline immunodeficiency virus ( fiv ), simian immunodeficiency virus ( siv ), human immunodeficiency virus type 1 ( hiv - 1 ), and human immunodeficiency virus type 2 ( hiv - 2 ) ( leis et al ., 1993 ). selective packaging of primer trna is defined as an increase in the percentage of the low molecular weight rna population representing primer trna in moving from the cytoplasm to the virus . for example , in amv , the relative concentration of trna trp changes from 1 . 4 % in the cytoplasm to 32 % in the virus ( waters et al ., 1977 ). in hiv - 1 produced from cos7 cells transfected with hiv - 1 proviral dna , both primer trna lys3 and trna lys1 , 2 are selectively packaged , and the relative concentration of trna lys changes from 5 - 6 % to 50 - 60 % ( mak et al ., 1994 ). both trna lys3 and trna lys1 , 2 are packaged into hiv - 1 with equal efficiency since the trna lys3 : trna lys1 , 2 ratio in the virus reflects the cytoplasmic ratio , even when the cytoplasmic ratio is altered ( huang et al ., 1994 ). in akr murine leukemia virus ( akr - mulv ), selective packaging of primer trna pro is less dramatic , going from a relative cytoplasmic concentration of 5 - 6 % to 12 - 24 % of low molecular weight rna ( waters et al ., 1977 ). selective packaging of primer trna occurs independently of viral genomic rna packaging in mulv , hiv - 1 , and avian sarcoma virus ( levin et al ., 1979 ; mak et al ., 1994 ; prats et al ., 1988 ), and has been shown in hiv - 1 to occur independently of gag and gag - pol processing ( khorchid et al ., 2000 ; mak et al ., 1994 ). selective packaging of primer trnas suggests that the increase in viral concentration of these trnas may facilitate the placement of the trna onto the pbs . this may be the case for avian retroviruses ( fu et al ., 1997 ; peters et al ., 1980 ) and hiv - 1 ( mak et al ., 1994 ), but is apparently not the case for mulv , where mutations in rt which prevent trna pro packaging do not inhibit its placement on the genome ( fu et al ., 1997 ; levin et al ., 1984 ; levin et al ., 1981 ). experiments with rt (−) mutants in avian retroviruses and in hiv - 1 do not make clear as to whether reduced genomic placement of primer trna is due to the reduction of primer trna in the virus or to the absence of functional rt sequences required to place the trna on the genome . however , recent experiments have shown that while pr160 gag - pol is required for selective packaging of trna lys3 into pr55 gag particles ( mak et al ., 1994 ), pr55 gag plays a major role in placing trna lys3 onto the pbs ( cen et al ., 1999 ; feng et al ., 1999 ). svc21bh10 is a simian virus 40 - based vector containing wild - type hiv - 1 proviral dna . svc21bh10 - lys3 and svc21 bh10 - lys2 contain both wild - type hiv - 1 proviral dna and a human trna lys3 or trna lys2 gene , respectively . these vectors were constructed as previously described ( huang et al ., 1994 ). cos7 cells were transfected using the calcium phosphate method as previously described ( mak et al ., 1994 ). supernatant was collected 63 hours post - transfection . for h9 , cemss , plb and u937 , an equal amount of infected and non - infected cells ( 5 × 10 6 cells each ) were mixed together , and supernatant containing virus was collected 3 days post - infection . virus from all cell types was pelleted from culture medium by centrifugation in a beckman ti45 rotor at 35 , 000 rpm for 1 hour . the viral pellets were then purified by centrifugation in a beckman sw41 rotor at 26 , 500 rpm for 1 hour through 15 % sucrose onto a 65 % sucrose cushion . the band of purified virus was removed and pelleted in 1 × tne in a beckman ti45 rotor at 40 , 000 rpm for 1 hour . viral genomic rna was extracted using guanidium isothiocynate , as previously described ( jiang et al ., 1993 ). electrophoresis of 32 pcp - labelled viral rna was carried out at 4 ° c . with the hoeffer se620 gel electrophoresis apparatus . the gel size was 14 by 32 cm . the first dimension was run in an 11 % polyacrylamide - 7m urea gel for 16 hours at 800 v . after autoradiography , the piece of gel containing rna was cut out , and run for 30 hours ( 25 watt limiting ); this was followed by autoradiography . all electrophoretic runs were carried out in 0 . 5 × tbe ( 1 × tbe is 50 mm tris , 5 mm boric acid , 1 mm edta - na 2 ). the electrophoretic gel patterns shown in this paper show only low molecular weight rna , since the high - molecular weight viral genomic rna cannot enter into the polyacrylamide gels . furthermore , these patterns represent only the most abundant trna species present , since longer film exposures will reveal the presence of more minor - abundance species . the relative amount of trna lys3 per copy of hiv - 1 genomic rna was determined by dot blot hybridization . each sample of total viral rna was blotted onto hybond n + l nylon membranes ( amersham pharmacia ) in triplicate , and was probed with a 5 ′ 32 p - end - labelled 18 - mer dna probe specific for the 3 ′ end of trna lys3 ( 5 ′- tggcgcccgmcagggac - 3 ′). the relative amounts of trna lys3 per sample were analyzed using phosphor - imaging ( biorad ). the blots were then stripped according to the manufacturer &# 39 ; s instructions , and were re - probed with a 5 ′ 32 p - end - labelled 17 - mer dna probe specific for the for the 5 ′ end of hiv - 1 genomic rna , upstream of the primer binding site ( 5 ′- ctgacgctctcgcaccc - 3 ′). phosphor - imaging was used to quantitate the relative amount of hiv - 1 genomic rna per sample , and the relative amount of trna lys3 per copy of hiv - 1 genomic rna was determined . trna lys3 - primed initiation of reverse transcription was measured by the ability of trna lys3 to be extended by 6 bases in an in vitro hiv - 1 reverse transcription reaction . for each sample , equal amounts of total viral rna ( 5 × 10 8 copies of genomic rna , measured as previously described ( huang et al ., 1994 )) were used as a source of primer trna / template . the sequence of the first 6 deoxynucleoside triphosphates incorporated is ctgcta . the reactions were carried out in a volume of 20 μl containing 50 mm tris - hcl ( ph 7 . 8 ), 100 mm kcl , 10 mm mgcl 2 , 10 mm dtt , 0 . 2 mm dctp , 0 . 2 mm dttp , 5 μci α - 32 p - dgtp and 0 . 05 mm ddatp ( instead of datp , thereby terminating the reaction at 6 bases ), 50 ng hiv - 1 rt , and rnase inhibitor ( amersham pharmacia ). after incubation for 15 minutes at 37 ° c ., the samples were precipitated with isopropanol , and were electrophoresed in a 6 % polyacrylamide gel at 70 w for 1 . 5 hours . relative amounts of trna lys3 placement were analyzed by comparing the intensity of bands with phosphor - imaging . viral infectivity was measured by the magi assay ( kimpton et al ., 1992 ). magi cells are cd4 + hela cells containing an hiv - 1 ltr fused to a β - galactosidase reporter gene . a total of 4 × 10 4 cells per well were cultured in 1 ml of media , in 24 - well plates . after 24 hours , the media was removed and was replaced with 150 μl of culture medium containing various dilutions of virus . deae - dextran was added to a final concentration of 20 μg / ml , and viral absorption took place for 2 hours , after which 1 ml of fresh culture medium was added . 48 hours later , the medium was removed and fixative ( 1 % formaldehyde , 0 . 2 % gluteraldehyde in pbs ) was added for 5 minutes . the fixative was removed and 200 μl of staining solution was added ( for 1 ml : 950 μl pbs , 20 μl of 0 . 2 m potassium ferrocyanide , 20 μl of 0 . 2 m potassium ferricyanide , 1 . 0 μl of 2 m mgcl 2 , and 10 μl of x - gal stock [ stock = 40 mg / ml in dmso ]). the cells were washed twice with pbs and the number of blue cells per well per equal amount of p24 were counted . viral particles were purified as described above , and viral proteins were extracted with ripa buffer ( 10 mm tris , ph 7 . 4 , 100 mm nacl , 1 % sodium deoxycholate , 0 . 1 % sds , 1 % np40 , 2 mg / ml aprotinin , 2 mg / ml leupeptin , 1 mg / ml pepstatin a , 100 mg / ml pmsf ). the viral lysates were analyzed by sds page ( 10 % acrylamide ), followed by blotting onto nitrocellulose membranes ( amersham pharmacia ). detection of protein by western blotting utilized monoclonal antibodies that are specifically reactive with hiv - 1 capsid ( zepto metrocs inc .) and reverse transcriptase ( a kind gift from m . parniak , montreal , canada ). detection of hiv proteins was performed by enhanced chemiluminescence ( nen life sciences products ) using sheep anti - mouse as a secondary antibody ( amersham life sciences ). effect of natural variation of trna lys3 packaging into hiv - 1 ( cos ) upon the initiation of reverse transcription and viral infectivity table 1a lists the trna lys3 / genomic rna ratio for 7 different preparations of hiv - 1 produced from cos7 cells . the values are normalized to the viral preparation containing the highest ratio , i . e . cos7a . each value listed is the average of experiments done in triplicate , in which dot blots of total viral rna were hybridized with radioactive dna probes complementary to either trna lys3 or genomic rna . it can be seen that within this sampling , the trna lys3 / genomic rna ratio can vary as much as three fold . three other viral preparations , cos7a , cos7b , and cos7c , are listed in table 1 b . normalizing against cos7b , the relative trna lys3 / genomic rna ratios are , respectively , 0 . 74 , 1 . 00 , and 0 . 52 . we have previously shown that alterations in the viral concentration of trna lys3 is reflected in opposite alterations in the viral concentration of trna lys1 , 2 , i . e ., an increase in the viral concentration of one isoacceptor results in a decrease in the viral concentration of the other isoacceptor ( feng et al ., 1999 ). 2 dimension polyacrylamide gel electrophoresis ( 2d page ) patterns of low molecular weight viral rna in these preparations , confirms this to be so . the identity of the trna lys isoacceptors found in each spot have been previously determined ( frugier et al ., 2000 ). analysis of the relative densities of each spot by phosphor - imaging gives the trna lys3 / trna lys1 , 2 ratio for each preparation . these are listed in table 1b , and it can be seen that they correlate with the trna lys3 / genomic rna ratios . the changes in viral trna lys3 concentrations are not as large as the corresponding changes in trna lys3 / trna lys1 , 2 ratios , because the ratios are determined by opposing changes in both trna lys3 and trna lys1 , 2 viral concentrations . we next investigated in these three viral preparations whether the amount of trna lys3 packaged into the virus reflects the amount of extendable trna lys3 placed onto the primer binding site ( pbs ). the first 6 bases incorporated into dna during the initiation of reverse transcription are ctgcta . trna lys3 extension was measured in an in vitro reaction using equal amounts of genomic rna , exogenous hiv - 1 rt , dctp , dttp , α - 32 p - dgtp , and ddatp . this will result in a six base extension of the trna lys3 , and the amount of dna extension / genomic rna was determined on 1 d - page ( data not shown ). relative signal intensities were measured by phosphor - imaging , the results of which are listed in table 1b . this data indicates a correlation between trna lys3 incorporated into the virus and the amount of extendable trna lys3 placed onto the pbs . the relative infectivity of the three viral preparations was also measured using the magi assay ( huang et al ., 1997 ), which measured single round infectivity . cd4 - positive hela cells containing the β - galactosidase gene fused to the hiv - 1 ltr are infected with virus . cells infected with hiv - 1 will have the β - galactosidase gene expressed , and such cells can be detected using an appropriate substrate for the enzyme , such as x - gal , whose metabolism turns the cells blue . the number of blue cells is a measure of viral infectivity . as indicated in table 1b , the relative infectivity of the different viral populations is directly correlated with trna lys3 packaging and extension . effect of artificially altering the trna lys3 concentration in hiv - 1 ( cos ) upon initiation of reverse transcription and viral infectivity . we have previously shown that viral trna lys3 content can be increased by transfecting cos7 cells with an sv40 - based plasmid containing both the hiv - 1 proviral dna and a human trna lys3 gene , and that as a result , trna lys1 , 2 packaging into the virus decreases ( huang et al ., 1994 ). herein , we have measured the effect of this artificial increase in viral trna lys3 ( virus bh 10 - lys3 in table 2 ) upon trna lys3 - primed initiation of reverse transcription and viral infectivity . we have also , in a similar manner , produced viruses with an excess of trna lys2 and a decrease in viral trna lys3 ( virus bh10 - lys2 in table 2 ), by transfecting cos7 cells with a plasmid containing the hiv - 1 proviral dna and a human gene for trna lys2 ( obtained from dr robert m . pirtle , university of north texas ). the relative concentration of trna lys3 / virion , normalized to wild type , was determined as above , by hybridizing dot blots of total viral rna with dna probes specific for trna lys3 and for genomic rna , and values are listed in table 2 . bh10 - lys3 has approximately 1 . 6 times more trna lys3 than wild type , while bh10 - lys2 has less than one fifth the amount of trna lys3 found in wild type virions . the 2d page pattern for low molecular weight rna in wild type hiv - 1 ( bh10 ), bh10 - lys3 , and bh10 - lys2 was assessed ( data not shown ), and the trna lys3 / trna lys1 , 2 ratios determined by phosphor - imaging of these gels are listed in table 2 . bh10 - lys3 has an additional small dark spot which has been identified as an additional trna lys3 by a partial t1 digestion pattern ( data not shown ) identical to the partial t1 digestion pattern of the major trna lys3 spot ( jiang et al ., 1993 ). this species can sometimes be seen as a very light spot in wild type virus . as found above for the different wild type hiv ( cos ), the changes in viral trna lys3 concentrations are not as large as the corresponding changes in trna lys3 / trna lys1 , 2 ratios because the ratios are determined by opposing changes in both trna lys3 and trna lys1 , 2 viral concentrations . as described above for wild type hiv ( cos ), we measured the ability of the placed trna lys3 from each viral preparation to be extended 6 bases in an in vitro reverse transcription reaction . the amount of trna lys3 extension / genomic rna was determined on 1 d - page , ( data not shown ). relative signal intensities were analyzed by phosphor - imaging , the results of which are listed in table 2 . this data indicates a direct correlation between trna lys3 incorporated into the virus and the amount of extendable trna lys3 placed onto the pbs . the relative infectivity of these different viral populations was also measured by the magi assay , and as indicated in table 2 , higher infectivity is associated with greater trna lys3 packaging and initiation of reverse transcription . while this data indicates that initiation of reverse transcription mirrors trna lys3 concentration in the virus , an alternative interpretation is that packaging and genomic placement of trna lys3 are both independently influenced by the packaging of pr160 gag - pol . we therefore looked at the rt / p24 ratios in bh10 - lys3 and bh10 - lys2 . a western blot of total viral protein from these two virus types probed with antibody to either p24 ( anti - ca ) or to rt ( anti - rt ) was carried out ( data not shown ). the ratio of rt / p24 , determined by phosphor - imaging , is 2 . 81 and 2 . 63 , respectively for bh10 - lys3 and bh10 - lys2 , making it unlikely that the five fold difference in placement of extendable trna lys3 between these two virus types is due to increased incorporation of pr160 gag - pol . effect of natural variation of trna lys3 packaging into hiv - 1 produced in chronically infected cell lines upon the initiation of reverse transcription and viral infectivity . the natural variation in trna lys3 packaging in hiv - 1 ( cos ) is also found in hiv - 1 produced in chronically infected cell lines . table 3a lists the trna lys3 / genomic rna ratio in hiv - 1 produced from 4 different chronically infected cell lines , and from transfected cos7 cells . two different viral preparations were used for each cell type , and the values were normalized to the the viral preparation containing the highest ratio , ie , cos7b . each value listed is the average of experiments done in triplicate , in which dot blots of total viral rna were hybridized with radioactive dna probes complementary to either trna lys3 or genomic rna . in table 3b , using different viral preparations , we measured the correlation between viral trna lys3 concentration , trna lys3 extension by rt , and viral infectivity , using methods described above for measuring these parameters in transfected cos7 cells . in table 3b , we see that the relative amount of trna lys3 extension and viral infectivity are directly correlated with the amount of viral trna lys3 packaging . to further understand the nature of the variation in trna lys3 packaging , we examined its stability in h9 cells chronically infected with hiv - 1 . every 3 days , cultures were supplemented with fresh uninfected h9 cells , keeping the cell concentration constant at 1 . 0 × 10 6 cells / ml , and viruses were harvested at 3 days , 2 weeks , one month , and 2 months . 2d page patterns of low molecular weight rna taken from these viruses enabled a determination of the ratios of trna lys3 / trna lys1 , 2 , phosphor - imaging . taken together , we have shown that increases in the ratio of trna lys3 / trna lys1 , 2 are correlated with increases in trna lys3 packaging into the virus and that over a two month period , the trna lys3 / trna lys1 , 2 changes , but that such changes are not stable . the work herein indicates a direct relationship between trna lys3 incorporated into the virus , trna lys3 - primed initiation of reverse transcription , and infectivity of the viral population . is placement proportional to the number of trna lys3 molecules within a virion , or are we simply recruiting new virions in the population that previously did not contain any trna lys3 ? the existence of defective viruses containing no trna lys3 is unlikely . we have shown that in a homogeneous population of hiv - 1 rt (−) mutants which are defective in selective trna lys packaging , there is still an average of 1 - 2 molecules trna lys3 packaged randomly per virion ( mak et al ., 1994 ; mak et al ., 1997 ). furthermore , trna lys3 extension in these rt mutant populations is defective ( 10 % wild type ( mak et al ., 1994 ) and unpublished results ). rather than 10 % of the defective viruses packaging all the trn lys , it seems more likely that all or nearly all virions in this defective rt (−) population contain 1 - 2 molecules of trna lys3 , and that this is not sufficient for correct placement of even one of the two pbs sequences present in each virion . if the increased trna lys3 packaging is accompanied by increased pr160 gag - pol incorporation , and if this viral protein is involved in packaging of trna lys into the virus , the increase in this protein could be responsible for greater trna lys3 placement . this seems unlikely for several reasons . first , we have previously shown that increased packaging of one trna lys isoacceptor family results in the reduction of the other trna lys isoacceptor family ( huang et al ., 1 . 994 ), something also seen by 2d - page analysis ( data not shown ). the total number of trna lys molecules in the virus does not change significantly , so there is no reason to assume that increased trna lys3 packaging is accompanied by an increased packaging of pr160 gag - pol . this was in fact demonstrated by western blots of protein from bh 10 - lys3 and bh10 - lys2 that the rt / p24 ratios are similar , even though trna lys3 extension in bh10 - lys2 is only 20 % that found in bh10 - lys3 ( data not shown ). secondly , work has shown , both in vitro ( feng et al ., 1999 ) and in vivo ( cen et al ., 1999 ), that the main viral protein involved in annealing trna lys3 to the pbs is pr55 gag , and not pr160 gag - pol . it is therefore likely that the inability of rt (−) mutants in avian retroviruses and hiv - 1 to place primer trna onto the pbs is due to the inability of these mutants to package primer trna , which does require intact rt sequence within pr160 gag - pol , and is not due to the absence of functional rt sequences in the virion . interestingly , this correlation between primer trna packaging and placement has not been found in rt (−) mulv , i . e ., rt (−) mutants which reduce packaging of primer trna pro do not reduce primer trna pro placement on the pbs ( fu et al ., 1997 ). since gag , rather than gag - pol , has been found to be sufficient for primer trna placement in vitro ( feng et al ., 1999 ), the insensitivity of genomic placement of primer trna in mulv to viral concentration of primer trna may reflect an increased binding affinity between murine gag and trna pro compared to the binding affinity between hiv - 1 gag and trna lys3 or avian gag and trna trp . this would also explain why the selective incorporation of primer trna in wild type virions is not required to be as strong in mulv as in avian retroviruses or hiv - 1 . the variation in trna lys3 packaged / virion that we report here was not previously seen in our earlier work with hiv - 1 - transfected cos cells ( huang et al ., 1994 ; mak et al ., 1997 ). what is responsible for the variability in the viral trna lys3 concentration ? since cultures of chronically infected cell lines are producing viruses which are constantly infecting uninfected cells , mutations in viral genes might occur over time during reverse transcription , and account for variability in trna lys packaging . however , since the variation in trna lys3 packaging is not stable this does not seem to be occurring . the inability of the virus to maintain higher levels of trna lys3 packaging , which we have shown to be associated with higher infectivity rates , also indicates that other constraints exist which must prevent viral mutations which would lead to higher trna lys3 packaging . cos3 cell transfection studies also indicate that the variability is not due to mutation in viral genes since reverse transcription is not involved in producing virions in this system . while the variability of trna lys3 packaging in such viruses could be due to errors arising during rna transcription , this would also seem unlikely to have a significant effect upon the whole population of first round viruses . the most likely explanation for the existence of unstable variation in the trna lys3 packaging is that it is due to an unstable variation in the cell environment . this could result in variations in the trna lys3 concentration in the cytoplasm , which previous work ( huang et al ., 1994 ) and the work herein with bh10 - lys3 and bh10 - lys2 have shown to have a direct effect upon the amount of trna lys3 packaged . the fact that variations detrimental to other events in the viral life cycle do not mask the increases in viral infectivity associated with increased trna lys3 packaging and placement indicate that the variation in trna lys3 packaging may represent a rather unique cellular event affecting viral infectivity , perhaps because trna lys is one of the few cellular factors known to be required in the viral life cycle . during hiv - 1 assembly , the major cellular trna lys isoacceptors , trna lys1 , 2 and trna lys3 are selectively packaged into the virus ( jiang et al ., 1993 ), and trna lys3 is used as the primer for the reverse transcriptase - catalyzed synthesis of minus strand dna ( leis et al ., 1993 ). the selective packaging of trna lys into hiv - 1 occurs independently of both genomic rna packaging ( jiang et al ., 1993 ) and the processing of the viral precursor proteins pr55 gag and pr160 gag - pol ( mak et al ., 1994 ), but does depend on the participation of both of these unprocessed proteins . while pr55 gag alone is sufficient to form viral particles , and binds to both viral genomic rna ( berkowitz et al ., 1996 ) and pr160 gag - pol ( park et al ., 1992 ; smith et al ., 1993 ), it is not known if a specific binding of pr55 gag to trna lys contributes to trna lys selective packaging . evidence for an interaction between pr55 gag and trna lys3 comes not from trna lys3 packaging studies , but from trna lys3 placement studies which indicate that this protein , and not pr160 gag - pol , plays a major role in annealing trna lys3 onto the pbs in vitro ( feng et al ., 1999 ) or in vivo ( cen et al ., 1999 ). in considering the interactions involved between viral proteins and trna lys during packaging , it must be taken into account that trnas have been reported to be channeled from one component of the translational machinery to the next , and thus , may never be free of this synthetic machinery ( stapulionis et al ., 1995 ). such components could involve ribosomes , elongation factors , and aminoacyl - trna synthetases ( aarss ). although it has been shown that elongation factor - 1 alpha is packaged into hiv - 1 via an interaction with pr55 gag ( cimarelli et al ., 1999 ), it is not clear how this protein , which binds to all aminoacylated trnas , would confer the ability to selectively package trna lys into the virion . another trna - binding protein in the cytoplasm which is more specific for trna lys is lysyl - trna synthetase ( lysrs ). this enzyme is an attractive candidate for interacting specifically with viral proteins , and may play a role in the transport of the three trna lys isoacceptors into the virions . we show herein that the trna lys - binding protein , lysyl - trna synthetase ( lysrs ), is also selectively packaged into hiv - 1 . the viral precursor protein pr55 gag alone will package lysrs into pr55 gag particles , independently of trna lys . with the additional presence of the viral precursor protein pr160 gag - pol , trna lys and lysrs are both packaged into the particle . while the predominant cytoplasmic lysrs has an apparent m r = 70 , 000 , viral lysrs associated with trna lys packaging is shorter , with an apparent m r = 63 , 000 . the truncation occurs independently of viral protease , and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer trna lys3 . svc21 . bh10 is a simian virus 40 - based vector that contains full - length wild - type hiv - 1 proviral dna and was a gift from e . cohen , university of montreal . psvgag - rre - r and psvfs5tprotd25g , which code for either gag or unprocessed gagpol , respectively , have been described previously ( smith et al ., 1990 ; smith et al ., 1993 ). viral production from either of these two plasmids , which contain the rev response element ( rre ), requires co - transfection with a rev protein expression vector , such as pcmv - rev . thus , co - transfection of psvgag - rre - r with pcmv - rev is required to produce virus - like particles containing unprocessed pr55 gag precursor protein . in this report , psvsf5tprotd25g is co - transfected with svc21p31l , a plasmid coding for hiv - 1 proteins including gag and rev , but not for stable gagpol . the construction of the mutants svc21dr2 , and svc21p31l have been described previously ( huang et al ., 1997 ; mak et al ., 1997 ). cos7 cells were maintained in dulbecco modified eagle medium with 10 % fetal bovine serum and antibiotic . h9 , plb , cemss and u937 cell lines (+/−, infected or non - infected ) were grown in rpmi1640 with 10 % fetal bovine serum and antibiotic . transfection of cos7 cells with the above plasmids by the calcium phosphate method was as previously described ( mishima et al ., 1995 ). viruses were isolated from cos7 cell culture medium 63 h posttransfection , or from the cell culture medium of infected cell lines . the virus - containing medium was first centrifuged in a beckman gs - 6r rotor at 3 , 000 rpm for 30 minutes and the supernatant was then filtered through a 0 . 2 μm filter . the viruses in the filtrate were then pelleted by centrifugation in a beckman ti45 rotor at 35 , 000 rpm for 1 h . the viral pellet was then purified by centrifugation with a beckman sw41 rotor at 26 , 500 rpm for 1 h through 15 % sucrose onto a 65 % sucrose cushion . sucrose - gradient - purified virions were resuspended in 1 × radioprecipitation assay buffer ( ripa buffer : 10 mm tris , ph 7 . 4 , 100 mm nacl , 1 % deoxycholate , 0 . 1 % sodium dodecyl sulfate ( sds ), 1 % nonidet p - 40 , protease inhibitor cocktail tablets ( boehringer mannheim )). western blot analysis was performed using either 300 μg of cellular protein or 10 μg of viral protein , as determined by the bradford assay ( bradford et al ., 1976 ). the cellular and viral lysates were resolved by sds - page followed by blotting onto nitrocellulose membranes ( gelman sciences ). detection of protein on the western blot utilized monoclonal antibodies or antisera specifically reactive with viral p24 and gp120 , as well as with different aminoacyl - trna synthetases . mouse anti - p24 and rabbit anti - gp120 were purchased from intracel corp . rabbit anti - lysrs , anti - prors , and anti - ilers were isolated following three subcutaneous injections of purified protein with 3 - 4 weeks intervals between injections ( 150 - 300 μg total protein ). an n - terminal truncated form of human lysrs ( shiba et al ., 1997 ), and a c - terminal truncated form of human ilers ( shiba et al ., 1994 ) were used in these preparations . the complete amino acid sequence of human lysrs can be found for example in shiba et al ., 1997 as well as in genbank under accession number d32053 . human prors is derived from the c - terminal domain ( amino acid residues 926 - 1440 ) of human glutamyl - prolyl - trna synthetase ( gluprors ), and was purified as described ( heacock et al ., 1996 ). western blots were analyzed by enhanced chemiluminescence ( ecl kit , amersham life sciences ) using goat anti - mouse or donkey anti - rabbit ( amersham life sciences ) as a secondary antibody . the sizes of the detected protein bands were estimated using pre - stained high molecular mass protein markers ( gibco / brl ). virions were sometimes purified by replacing centrifugation through sucrose with centrifugation in an optiprep velocity gradient ( 60 % [ wt / vol ] iodixanol , life technologies ). iodixanol gradients were prepared in pbs as 11 steps in 1 . 2 % increments ranging from 6 to 18 %. virions were layered onto the top of the gradient and centrifuged for 1 . 5 h at 26 , 500 rpm in a beckman sw41 rotor . fractions were collected from the top of the gradient . aliquots were resuspended in pbs and centrifuged for 1 h at 40 , 000 rpm in a beckman ti50 . 3 rotor . the resulting pellets were resuspended in ripa buffer and resolved using sds - page , followed by either coomassie blue staining or western blot analysis . subtilisin digestion assays were performed essentially according to ott et al ( ott et al ., 1995 ). the purified virions were mock treated or treated with 1 mg / ml of subtilisin ( boehringer mannheim ) in digestion buffer ( 10 mm tris - hcl , ph 8 , 1 mm cacl 2 and bsa ) for 16 h at 37 ° c . subtilisin was inactivated by phenylmethylsufonyl fluoride . virions were then repelleted , resuspended in 2 × loading buffer ( 120 mm tris - hcl , ph6 . 8 , 20 % glycerol , 4 % sds , 200 mm dtt , 0 . 002 % w / v bromephenol blue ) and subjected to sds page , followed by western blot analysis , using anti - lysrs , anti - p24 and anti - gp120 . his 6 - tagged full length human lysrs was overexpressed in escherichia coli , and purified as previously described ( shiba et al ., 1997 ). [ 0193 ] fig1 a shows western blots of some aminoacyl - trna synthetases found in the cytoplasm of cos7 cells transfected with hiv - 1 and in the viruses produced . panel a represents a western blot of either viral ( v ) or cytoplasmic ( c ) proteins probed with an antibody to human lysrs . in both the cos cell cytoplasm and in the viruses , lysrs species can be detected in three sizes . the apparent molecular weights ( m r &# 39 ; s ) of these peptides , determined by sds page ( fig1 and 3 ), are 70 , 000 for the large species , 63 , 000 for the intermediate species , and 62 , 000 for the small species . the large species predominate in the cytoplasm , while in the virus , both large and intermediate species are present . the sizes of the lysrs species determined by sds page are only approximate sizes since the calculated size of the human lysrs coded by a full length lysrs cdna is 597 aa protein , with an m r of 68 , 034 ( shiba et al ., 1997 ). [ 0194 ] fig1 a also shows western blots of cytoplasmic or viral protein probed with antibodies to human isoleucyl - trna synthetase ( ilers ) ( panel b ) or human prolyl - trna synthetase ( prors ) ( panel c ). human ilers contains 1266 amino acid residues , with an m r of approximately 152 , 000 ( shiba et al ., 1994 ). in all higher eukaryotes examined , prors is the c - terminal part of a fusion with glurs ( cerini et al ., 1991 ; heacock et al ., 1996 ), while the purified prors has an m r of approximately 60 , 000 ( ting et al ., 1992 ). while these proteins are detected in the cytoplasm , they are not detected in the viruses , indicating that incorporation of lysrs into viruses is non - random . the presence of lysrs within the virus is further substantiated by its resistance to digestion by the protease subtilisin ( fig1 b ). intact viruses were either untreated ( n ) or treated with subtilisin ( s ) before viral lysis , and western blots were probed with anti - p24 ( panel a ), anti - gp120 ( panel b ), and anti - lysrs ( panel c ). the results show that p24 , pr55 gag , and lysrs are resistant to proteolysis , while external proteins gp160 and gp120 are susceptible to proteolysis by subtilisin . this indicates that lysrs is present within the virus . lane k contains purified , his 6 - tagged human lysrs , which in panel c has not been exposed to protease . however , exposure of this purified protein to subtilisin does degrade it ( panel d ). the his 6 - tagged human lysrs migrates more slowly than the large cytoplasmic lysrs species because of the n - terminal mrgshhhhhhssgwvd sequence appended to the full - length human lysrs used in these studies ( shiba et al ., 1994 ). the virions studied in this work are purified by centrifugation through 15 % sucrose to the surface of a 65 % sucrose cushion . to further confirm that these viruses do not contain contaminating lysrs bound to their surface , viruses were also purified using velocity centrifugation through a 6 - 18 % iodixanol gradient ( optiprep , nycomed pharma , norway ) instead of centrifugation through sucrose . optiprep gradients have been shown to produce viruses more free from cytoplasmic contaminants than obtained using sucrose gradients ( dettenhofer et al ., 1999 ). fig2 shows western blots of gradient fractions probed with anti - p24 ( panel a ) and anti - lysrs ( panel b ) following optiprep gradient purification . we observe that lysrs comigrates with the viral pr55 gag protein . panel c shows the different gradient fractions stained with coomassie blue , and indicates that most residual cellular protein is found in fractions closer to the top of the gradient rather than where viral protein and lysrs migrate . twenty times more viral lysate than used in panels a and b was used to visualize the proteins by commassie blue staining . although the lysrs is detected in the same optiprepl gradient fractions as p24 , the lysrs / p24 ratio is much smaller in the heavier fractions 1 and 2 than in fractions 3 - 5 . the bottom - most fractions could represent aggregates of broken virus no longer containing lysrs , or the anti - lysrs may have a lower sensitivity than anti - p24 . in panel d , cell culture medium from non - transfected cos7 cells was resolved in the optiprep gradient , and probing with anti - lysrs shows the absence of lysrs in the medium . sizes of lysrs incorporated into hiv - 1 produced from transfected cos7 cells and chronically - infected cell lines although both large and intermediate size lysrs species are found in hiv - 1 produced from cos7 cells , the intermediate size peptide is the major lysrs found in hiv - 1 produced from chronically infected cell lines . this is shown in the western blots probed with anti - lysrs in fig3 . in the cytoplasm of h9 cells , uninfected ( lane 10 ) or chronically - infected with hiv - 1 ( lane 9 ), the major lysrs species is the large species , with a small amount of small species also present . similar results are also found in the cytoplasm of plb , cemss and u937 cells ( data not shown ). on the other hand , in virions produced from these four chronically infected cell lines , the major lysrs species packaged is the intermediate size lysrs species . the ratio of intermediate to large lysrs species found in hiv ( cos ) can be influenced by the amount of trna lys3 synthesized in the cell and packaged into the virion . it has already been shown that transfection of cos cells with a vector containing both hiv - 1 proviral dna and a trna lys3 gene , results in an increase in trna lys3 in the cytoplasm and in the virus ( huang et al ., 1994 ). in fig4 the effect of excess trna lys3 on the level of lysrs in the cytoplasm and in the virus was analyzed . cos cells were transfected with either wild type ( wt ) hiv - 1 proviral dna or a plasmid containing both wt hiv - 1 proviral dna and a trna lys3 gene . the amount of p24 present in each viral preparation was determined by western blot of viral protein probed with anti - p24 ( see panel a ). in panel b , viral protein containing equal amounts of p24 were blotted and probed with anti - lysrs ( lanes 1 and 2 ). it can be seen that virions produced from cells with excess trna lys3 also contain an excess of the intermediate species of lysrs . indeed , densitometry analysis indicated that there was a 3 - fold increase in the intermediate lysrs species as compared to that found in wild type viruses . the presence of the intermediate form of lysrs is also increased in the cytoplasm of these cells ( see lanes 3 and 4 in panel b ). taken together , such results show a positive correlation between the quantity of trna lys3 and that of lysrs inside the virions . mutant viruses previously shown to be deficient in trna lys incorporation ( huang et al ., 1997 ; mak et al ., 1997 ) were produced by transfecting cos7 cells with wild type and mutant hiv - 1 proviral dna , and the incorporation of lysrs into the virions was analyzed by western blots , as shown in fig5 . lanes 1 and 7 show purified his 6 - tagged - lysrs and lysrs found in cos7 cell cytoplasm , respectively . lanes 2 and 3 represent protein from wild - type ( wt ) or protease - negative ( pr (−)) viruses , respectively . both viruses have been shown to selectively incorporate trna lys ( jiang et al ., 1993 ; khorchid et al ., 2000 ), and lanes 2 and 3 show they both contain the large and intermediate size species of lysrs . lanes 4 - 6 represent western blots of protein from mutant viral - like particles ( vlps ) p31l , dr2 , and pr55 gag , none of which incorporate either pr160 gag - pol or trna lys ( huang et al ., 1997 ; khorchid et al ., 2000 ; mak et al ., 1994 ; mak et al ., 1997 ). p31l contains a substitution of p for l at position 31 in nucleocapsid protein ( ncp7 ) in the basic amino acid sequence between the two cys - his boxes . this mutation causes the rapid degradation of p160 gag - pol in the cytoplasm ( huang et al ., 1997 ). dr2 is a substitution mutation in the connection domain of rt , in which f 389 is replaced with f 389 ag , and also causes the rapid degradation of pr160 gag - pol in the cytoplasm ( mak et al ., 1997 ). lane 6 represents protein from pr55 gag vlps produced by transfecting cos cells with the vector psvgag - rre , which codes only for pr55 gag ( smith et al ., 1993 ). these three different vlps , which do not selectively package trna lys , do not contain the intermediate size lysrs species , but do contain the large and small species of lysrs . thus , the incorporation of lysrs into viral particles appears dependent upon pr55 gag protein , and is independent of trna lys or pr160 gag - pol incorporation . however , the presence of intermediate size lysrs in viruses appears to be directly correlated with the packaging of trna lys and pr160 gag - pol . we have previously reported that selective packaging of trna lys can be partially rescued in the p31l vlp by cotransfection of cos cells with p31l proviral dna and dna coding for wild type pr160 gag - pol , but not with dna coding for wild - type pr55 gag ( huang et al ., 1997 ). the effect of the rescue of trna lys packaging upon lysrs incorporation was investigated next . fig5 b shows a western blot probed with anti - lysrs , containing purified his 6 - tagged lysrs ( lane 1 ), and protein from protease - negative hiv - 1 , which packages trna lys and which shows the large and intermediate size lysrs species ( lane 2 ). lane 3 contains protein from the p31l mutant , which does not package trna lys , pr160 gag - pol , or the intermediate size lysrs . cotransfection with psvfs5tprotd25g , which codes for wild type pr160 gag - pol , and which partially rescues trna lys packaging , also results in a small amount of intermediate size lysrs incorporation ( lane 4 ). in contrast , cotransfection with psvgag - rre - r , which codes for wild type pr55 gag , and which does not rescue trna lys packaging , also does not result in the incorporation of intermediate size lysrs ( lane 5 ). in this work , we have provided evidence for the incorporation of human lysrs into hiv - 1 . this evidence included detection of lysrs in virions purified by centrifugation using either sucrose or optiprep gradients . two other human aminoacyl - trna synthetases , prors and ilers , were not detected in virions , though they were readily detected in the cytoplasm of hiv - 1 - transfected cells . while purified lysrs was susceptible to degradation by the protease subtilisin , lysrs detected in viruses was resistant to subtilisin digestion under reaction conditions in which external envelope protein gp120 was degraded . we detect lysrs in three sizes , with apparent molecular weights on sds gels of 70 , 000 ( large species ), 63000 ( intermediate species ), and 62 , 000 ( small species ). the results in fig5 indicate that pr55 gag alone among the viral proteins is sufficient for incorporating lysrs . the gag vlps do not incorporate either trna lys or pr160 gag - pol , and the intermediate lysrs is replaced with the small species . the three types of pr55 gag vlps ( fig5 a , lanes 4 - 6 ) do not incorporate either trna lys or pr160 gag - pol . the viral - like particles which contain only pr55 gag ( fig5 a , lane 6 ) are produced by cotransfecting cells with psvgag - rre - r and pcmv - rev . the hiv - 1 proviral dna in the former plasmid not only lacks viral sequences downstream of gag ( except for the rre ), but an sv40 late promoter region has replaced all viral sequences upstream of nucleotide 679 in the viral dna . the viral - like particles produced are defective in incorporating the truncated genomic rna as well as trna lys and pr160 gag - pol ( mak et al ., 1994 ; smith et al ., 1990 ; smith et al ., 1993 ). pr55 gag may interact with a cytoplasmic trna lys / lysrs complex and destabilize it , thereby releasing the trna lys and resulting in the incorporation of lysrs alone into the gag vlp . the additional presence of pr160 gag - pol may serve to stabilize the pr55 gag / trna lys / lysrs ternary complex since pr160 gag - pol interacts with both trna lys ( khorchid et al ., 2000 ) and pr55 gag ( park et al ., 1992 ; smith et al ., 1993 ). destabilization of the lysrs / trna lys complex by the large number of pr55 gag molecules in the cell might be expected to inhibit translation . there are a number of possible reasons why this does not happen . most pr55 gag molecules may not bind lysrs , either because pr55 gag molecules without pr160 gag - pol have a weaker affinity for lysrs , or because pr55 gag only interacts with lysrs as a multimeric pr55 gag complex . additionally , the destabilization of trna lys / lysrs may release free non - acylated trna lys , a molecule which has been shown in yeast to induce the synthesis of more lysrs ( lanker et al ., 1992 ), which could help maintain the cytoplasmic concentrations of trna lys / lysrs and lysine - trna lys required for translation . we do not yet know if pr55 gag interacts directly with lysrs . since the plasmid coding for the pr55 gag protein , psvgag - rre - r , codes only for this protein , ( smith et al ., 1990 ), vpr , a viral protein which was shown to interact with human lysrs both in vitro and in the yeast two hybrid system ( stark et al ., 1998 ), is not needed for the incorporation of lysrs into the pr55 gag particles . we have also previously shown that trna lys is selectively incorporated into hiv - 1 missing vpr ( khorchid et al ., 2000 ). on the other hand , pr55 gag might interact indirectly with lysrs via another cellular trna - binding protein , such as elongation factor 1 - alpha , which has been shown to interact with pr55 gag and to be incorporated into hiv - 1 during assembly ( cimarelli et al ., 1999 ). the dominant lysrs form in viruses produced from the human cell lines is the intermediate form ( fig3 ). since truncation of lysrs to the small species also occurs in gag vlps , processing does not depend upon the presence of either pr160 gag - pol or trna lys , but may be limited by them to produce the intermediate species . the predominance of large lysrs in the cytoplasm and intermediate lysrs in the viruses ( particularly in viruses produced from human cell lines ) suggests that the intermediate and small lysrs species may be generated by proteolysis of the large species , a phenomenon observed during the in vitro proteolytic cleavage of the n terminal regions of dimeric yeast ( ciracoglu et al ., 1985 ) or sheep ( cirakoglu et al ., 1985 ) lysrs to truncated homodimers . the detection of lysrs heterodimers in sheep has also been reported ( cirakoglu et al ., 1985 ). however , if a protease is involved , it is not a viral protease since processing of lysrs occurs in both gag vlps and in protease - negative virions . a recent report does indicate that the human cytoplasmic and mitochondrial lysrss are generated by alternative splicing of the same primary rna transcript ( tolkunova et al ., 2000 ). the mitochondrial lysrs contains extra amino acid sequences used for mitochondrial targeting in the n - terminal region , and because it is larger than the cytoplasmic lysrs , it is unlikely to be represented by the intermediate and small species observed in the present studies . alternate rna splicing has also been reported for generating human cytoplasmic cysteinyl - trna synthetase ( kim et al ., 2000 ). very little processed lysrs is detected in the cytoplasm of chronically - infected cell lines ( fig3 ), and this is the small species . these data presented herein therefore appear to support the possibility that the processing of the large lysrs species to the intermediate species occurs during or after viral release from the cell . we cannot exclude the possibilities that either non - detectable amounts of intermediate lysrs in the cytoplasm are selectively packaged into the virus , or that the scarcity of the intermediate lysrs species in the cytoplasm is due to the fact that it is selectively packaged into the virus . the presence of both large and intermediate species of lysrs in hiv - 1 produced from cos7 cells does indicate that the large species is capable of being packaged into the virion , however . while the ratio of intermediate to large lysrs species varies from one preparation of hiv ( cos ) to the next ( for example , compare fig1 c with fig1 a or 4 b ), it is usually greater than 1 and increases when trna lys3 packaging increases ( fig4 b ). it has been shown that removal of n - terminal sequence from yeast asprs weakens binding of the enzyme to the trna asp , as shown by an increase in both the kd for trna binding and k m of the aminoacylation reaction of approximately 2 orders of magnitude ( frugier et al ., 2000 ). on the other hand , human lysrs missing the n - terminal 65 amino acids did not display significantly reduced in vitro aminoacylation kinetics ( shiba et al ., 1994 ), implying a similar trna lys binding affinity as for wild type lysrs . of note , the removal of the n - terminal extension of human lysrs , absent in prokaryotic enzymes , was shown to be dispensable for its in vitro aminoacylation activity and for the in vivo cross - species complementation from human to e . coli ( shiba et al ., 1997 ). reduced affinity of the intermediate lysrs for trna lys might therefore be due to other lysrs sequences missing , or to a cellular environment different from that tested in vitro . regulation of trna lys incorporation into hiv - 1 by lysyl trna synthetase we have shown that during hiv - 1 assembly in cos7 cells transfected with hiv - 1 proviral dna , lysyl trna synthetase ( lysrs ) and the major trna lys isoacceptors , trna lys1 , 2 and trna lys3 , are selectively packaged into the viruses . pr55 gag alone is sufficient for packaging lysrs into pr55 gag particles , but the additional presence of pr160 gag - pol is required for trna lys incorporation as well . since pr160 gag - pol interacts with both pr55 gag ( park et al ., 1992 ; smith et al ., 1990 ) and trna lys ( khorchid et al ., 2000 ; mak et al ., 1994 ), its presence may stabilize the pr55 gag / lysrs / trna lys complex . it is not known if pr55 gag interacts directly with lysrs or through another cellular trna - binding protein , such as elongation factor 1 - alpha ( ef1 ∀). ef1 ∀ has been shown to interact directly with pr55 gag and to be incorporated into hiv - 1 during assembly ( cimarelli et al ., 1999 ) on the other hand , vpr , a viral protein which was shown to interact with human lysrs in vitro and in the yeast two hybrid system ( stark et al ., 1998 ), is not needed for the incorporation of lysrs into the pr55 gag particles , since plasmids used to produce pr55 gag viral - like particles which package lysrs did not code for vpr ( example 2 ), and trna lys is also selectively incorporated into vpr - negative hiv - 1 ( mak et al ., 1994 ). whether vpr plays another role , such as in facilitating trna lys3 genomic placement or deacylating trna lys3 , is not yet known . in the cytoplasm of uninfected or infected cells , sds page indicates that there exists both an abundant lysrs species with an apparent molecular weight of approximately 68 , 000 ( large species ), and a smaller less abundant species with an approximate molecular weight of 62 , 000 ( small species ). in hiv - 1 produced from a number of cell lines , the predominant lysrs species has an intermediate molecular weight of 63 , 000 ( example 2 ). in hiv - 1 produced from cos7 cells , both large and intermediate lysrs species are present , usually in similar amounts . the intermediate species is always present in viruses incorporating trna lys . the production of virus - like particles ( vlps ) composed only of pr55 gag is sufficient for incorporation of lysrs . however , trna lys is not selectively packaged into these particles , and only the large and intermediate lysrs species are present in the viruses . since the intermediate species is present in protease - negative viruses , and the small species in pr55 gag vlps ( example 2 ), the intermediate and small species could not be generated by a viral protease . the precise nature of the modification of these lysrs species is not yet known , and appears to be due to a cellular protease ( cirakoglu et al ., 1985 ). alternatively , it could be due to alternative splicing of the same primary rna transcript ( tolkunova et al ., 2000 ). lysrs truncation could result in weakening the interaction between lysrs and trna lys ( frugier et al ., 2000 ) which might facilitate either trna lys interaction with viral proteins during packaging or annealing to the viral genomic rna . herein , it is shown that trna lys packaging is limited by the level of lysrs , since the overproduction of lysrs from a cotransfected plasmid encoding lysrs results in up to a 2 fold increase in the incorporation of both trna lys isoacceptors into the viruses . overproduction of lysrs also results in an increase in both lysrs packaging into hiv - 1 and in the cytoplasmic concentrations of both trna lys isoacceptors . however , increased cytoplasmic concentrations of trna lys are not the prime cause of increased trna lys incorporation into viruses . overproduction of a mutant lysrs lacking the n terminal 65 amino acids also results in increases in both lysrs viral packaging and trna lys concentrations in the cytoplasm , but no increase in trna lys viral packaging is observed . this probably reflects the weaker affinity the mutant lysrs has for trna lys , as demonstrated by electrophoretic band shift assays of in vitro trna lys3 / lysrs binding . wild type lysrs can migrate to the nucleus , but since the n - terminal mutant lysrs has lost this ability , increased trna lys gene expression is not due to a direct stimulation of transcription or nuclear export by lysrs . svc21 . bh10 p - is a simian virus 40 - based vector that contains full - length wild - type hiv - 1 proviral dna containing an inactive viral protease ( d25g ), and obtained from e . cohen , university of montreal . pm368 contained cdna encoding full length ( 1 - 597 amino acids ) human lysrs , was obtained from shiba et al ., 1997 . the cdna was pcr - amplified , and digested with ecor1 and xho1 , whose sites were placed in each of the pcr primers . to produce an n - terminal truncated lysrs encoding amino acids 66 - 597 , the sense primer was complementary to a downstream sequence . for expression in cos7 cells , the pcr dna fragments were cloned into either pcdna 3 . 1 ( invitrogen ) to obtain plysrs . f and plysrs . t , expressing full length or n - terminal truncated lysrs , respectively , or into pcdna3 . 1 / v5 - hisa , which adds c - terminal tags v5 and his 6 to the wild type ( lysrs . cf ) and mutant ( lysrs . ct ) lysrs species . to purify the wild type and mutant lysrs , the pcr dna fragments were cloned into the bacterial expression vector pet - 21b (+) ( clonetech ), which expresses the proteins with a c - terminal his 6 tag . his 6 - tagged full length and truncated human lysrs was overexpressed in escherichia coli , and purified as previously described ( shiba et al ., 1997 ). cos7 cells were maintained in dulbecco modified eagle medium with 10 % fetal bovine serum and antibiotic . for cell fractionation , cells were resuspended in lysis buffer ( pbs with 0 . 1 % nonidet p - 40 , 0 . 1 % triton x - 100 , and protease inhibitor coctail tablets ( roche )), and incubated on ice for 10 minutes . nuclei were pelleted by centrifugation at 1000 × g for 10 minutes at 4ec , and the supernatant was collected as the cytoplasmic fraction . nuclear extracts were prepared by lysing nuclei in ripa buffer . western blot analysis of the total cell lysate , postnuclear supernatant and nuclear extracts were performed as described below , using anti v5 ( invitrogen ), anti - tubulin ( santa cruz biotechnology ) and anti - yyi ( santa cruz biotechnology ). anti - v5 was used to detect lysrs . cf and lysrs . ct , wild type and mutant lysrs which contain a c - terminal 14 amino acid v5 epitope . transfection of cos7 cells with the above plasmids by the calcium phosphate method was as previously described ( mak et al ., 1994 ). viruses were isolated from cos7 cell culture medium 63 h posttransfection , or from the cell culture medium of infected cell lines . the virus - containing medium was first centrifuged in a beckman gs - 6r rotor at 3 , 000 rpm for 30 minutes and the supernatant was then filtered through a 0 . 2 μm filter . the viruses in the filtrate were then pelleted by centrifugation in a beckman ti45 rotor at 35 , 000 rpm for 1 h . the viral pellet was then purified by centrifugation with a beckman sw41 rotor at 26 , 500 rpm for 1 h through 15 % sucrose onto a 65 % sucrose cushion . total cellular or viral rna was extracted from cell or viral pellets by the guanidinium isothiocyanate procedure ( chomczynski et al ., 1987 ), and dissolved in 5 mm tris buffer , ph 7 . 5 . hybridization to dot blots of cellular or viral rna were hybridized with dna probes complementary to trna lys3 and trna lys1 , 2 ( jiang et al ., 1993 ), genomic rna ( cen et al ., 1999 ), and 3 - actin mrna ( dna probe from ambion ). 2d page of 32 pcp - 3 ′ end labeled viral rna was carried out as previously described ( jiang et al ., 1993 ). sucrose - gradient - purified virions were resuspended in 1 × radioprecipitation assay buffer ( ripa buffer : 10 mm tris , ph 7 . 4 , 100 mm nacl , 1 % deoxycholate , 0 . 1 % sodium dodecyl sulfate ( sds ), 1 % nonidet p - 40 , protease inhibitor cocktail tablets ( boehringer mannheim )). western blot analysis was performed using either 300 μg of cellular protein or 10 μg of viral protein , as determined by the bradford assay ( bradford et al ., 1976 ). the cellular and viral lysates were resolved by sds polyacrylamide gel electrophoresis ( sds - page ), followed by blotting onto nitrocellulose membranes ( gelman sciences ). detection of protein on the western blot utilized monoclonal antibodies or antisera specifically reactive with viral p24 ( mouse antibody , intracel ), 3 - actin ( sigma aldrich ), and human lysrs ( rabbit antibody , obtained from k . shiba ( shiba et al ., 1997 )). western blots were analyzed by enhanced chemiluminescence ( ecl kit , amersham life sciences ) using goat anti - mouse or donkey anti - rabbit ( amersham life sciences ) as a secondary antibody . the sizes of the detected protein bands were estimated using pre - stained high molecular mass protein markers ( gibco / brl ). trna lys was purified from human placenta as previously described ( jiang et al ., 1993 ), and labeled with the 3 ′- 32 pcp end - labeling technique as previously described ( bruce et al ., 1978 ). in 20 : 1 binding buffer ( 20 mm tris - hcl , ph 7 . 4 , 75 mm kcl , 10 mm mgcl 2 , and 5 % glycerol ) 5 nm labeled trna lys was incubated with different concentrations of lysrs ( 0 . 06 um , 0 . 3 um , or 1 . 5 . um ) for 15 minutes on ice , and then analyzed by 1 d - page ( native 6 % gels in 1 × tbe at 4 ° c .). lysrs overexpression : effect upon cytoplasmic and viral concentrations of lysrs and trna lys isoacceptors cos7 cells were transfected with plasmids coding for either full length lysrs ( lysrs . f ) or a truncated lysrs , in which the first n - terminal 65 amino acids have been deleted ( lysrs . t ). fig7 shows western blots of cell lysates , probed with either anti - lysrs ( panel a ) or anti - actin ( b ). the bands were quantitated by phosphorimaging , and the lysrs / actin ratios are shown in panel c , normalized to the lysrs / actin in non - transfected cos7 cells . lysrs . t is expressed somewhat better than lysrs . f , as shown by the higher lysrs / actin ratios . these ratios are similar in cells without viruses ( lanes 1 - 3 ) and in cells producing viruses ( lanes 4 - 6 ). as shown in fig8 the overexpression of lysrs in cells cotransfected with a plasmid ( bh10p -) containing protease - negative hiv - 1 proviral dna results in increased packaging of lysrs in the viruses produced . fig8 a shows western blots of viral lysates probed with either anti - lysrs or anti - ca . as previously reported ( example 2 ), lysrs in virions produced from cos7 cells contains both the full length ( large ) lysrs , and the intermediate species . the bands were quantitated by phosphorimaging , and the lysrs / gag ratios are shown in panel c , normalized to the lys / gag ratio for cells transfected with bh 10p - only ( lane 1 ). it can be seen that more lysrs . t is incorporated into virions than lysrs . f , which may reflect the higher amount of lysrs . t present in the cytoplasm . this reduced overexpression of lysrs . f compared to lysrs . t may be due the ability of lysrs . f to feedback - inhibit its own synthesis by returning to the nucleus , something lysrs . t cannot do ( see below and fig1 ). [ 0231 ] fig9 shows the effect of lysrs overexpression upon trna lys concentrations in the cytoplasm of hiv - 1 - transfected - cos7 cells and in the virions produced from these cells . dot blot , hybridization was used to determine the trna lys3 / actin mrna and trna lys1 , 2 / actin mrna ratios in total cytoplasmic rna , using hybridization probes specific for these rnas ( jiang et al . 1993 ). the results , quantitated by phosphorimaging , are shown in fig9 a . small increases in the cytoplasmic concentrations of the major trna lys isoacceptors are seen using either lysrs . f or lysrs . t . in the experiments represented in panels b , dot blot hybridization of total rna isolated from the virus produced in these cells was used to measure the trna lys3 / genomic rna and the trna lys1 , 2 / genomic rna ratios . the results , quantitated by phosphorimaging , are shown in panel b . it is quite clear that only the overexpression of lysrs . f results in an increase in the incorporation of trna lys isoacceptors into the viruses . this result can also be seen in panel c , which shows the resolution of viral trna lys isoacceptors by 2d - page . total viral rna samples containing equal amounts of genomic rna were end - labeled with 32 pcp , and the low molecular - weight rna , which is the only rna able to enter the gel , was resolved by 2d - page . the position of the trna lys isoacceptors is as previously determined ( jiang et al ., 1993 ), and while the ratio of these isoacceptors to each other in the viruses does not change , it is clear that on a genomic rna basis , the trnas in virions produced from cells overexpressing lysrs . f show the strongest signal , thereby supporting the conclusions derived from the dot blot hybridiation data in panel b , ie , overexpression of lysrs . t does not induce greater packaging of trna lys isoacceptors into the viruses . the slowest moving trna species , “ 4 ”, is not a trna lys isoacceptor , and has been tentatively identified as trna asn ( data not shown ), a trna species previously reported to be packaged into hiv - 1 ( zhang et al ., 1996 ). the spot 4 : trna lys ratio appears to decrease upon expression of excess lysrs . f , but increases upon expression of lysrs . t . lysrs . t binds more weakly to trna lys3 in vitro than lysrs . f overexpression of lysrs . t results in similar increases in both the cytoplasmic concentrations of trna lys and in viral incorporation of lysrs , yet , unlike lysrs . f , does not result in greater trna lys packaging into the virion . one explanation may be that lysrs . t cannot bind as well to trna lys isoacceptors as lysrs . f , and we have investigated this . n - terminal , his - tagged human lysrs , wild type or mutant , was purified by ni ++ chromatography ( shiba et al ., 1997 ), and human trna lys3 was purified from human placenta , as previously described ( jiang et al ., 1993 ). the ability of lysrs to bind radioactive trna lys3 in vitro was determined using an electrophoretic band shift assay , and the results are shown in fig1 . human trna lys3 was 3 - end labeled with 32 pcp ( bruce et al ., 1978 ), and incubated with increasing amounts of purified lysrs . t or lysrs . f . the resulting complexes were resolved on 1d - page , and fig1 indicates that lysrs . f has a greater ability to form complexes with the labeled trna lys3 than does lysrs . t . because overexpression of either lysrs . f or lysrs . t results in an increase in the cytoplasmic concentrations of trna lys isoacceptors , we investigated the possibility of a direct derepression of trna lys genes by lysrs as a result of lysrs migrating into the nucleus . cos7 cells were transfected with plasmids coding for either lysrs . cf or lysrs . ct , where the “ c ” indicates that the lysrs has been c - terminally tagged with the 14 amino acid v5 epitope . cells were lysed in 0 . 1 % np - 40 , and western blots were used to examine either the total lysate ( t ), or cell lysate fractionated by low speed centrifugation , into nuclear ( n ) and cytoplasm ( c ) compartments . fig1 a shows the distribution of wild type and mutant lysrs in the cell . the first 3 lanes detect endogenous lysrs in non - transfected cells , using anti - lysrs , and show that while both the full length and smaller lysrs appear in the total lysate and in the cytoplasm , as previously described ( example 2 ), only the full length lysrs can be seen in the nuclear fraction . the next 6 lanes use anti - v5 to detect exogenous full length ( lysrs . cf ) and experimentally truncated lysrs ( lysrs . ct ) in the different cell fractions . the expression of lysrs . cf in the cell does result in the generation of some smaller peptides in the cytoplasm , but clearly only the full length lysrs . cf is seen in the nucleus . since the smaller fragments must contain the c - terminal tag v5 to be detected by anti - v5 , the smaller fragments may have resulted from n - termnal deletions . in fact , as shown in the last three lanes , experimental deletion of the n - terminal 65 amino acids ( lysrs . ct ) results in the inability of this truncated lysrs to migrate to the nucleus . panels b and c represent controls for the purity of the nuclear and cytoplasmic preparations , ie , the known cytoplasmic protein , alpha tubulin , is not detected in the nuclear fraction ( panel b ), while the nuclear transcription factor , yyi , is primarily found in the nucleus ( panel c ). increasing the cytoplasmic concentration of wild type lysrs in cos7 cells by transfecting cells with lysrs . f results in an approximately 20 % increase in the cytoplasmic concentration of trna lys and an approximately 2 fold increase in the incorporation of trna lys3 and trna lys1 , 2 into virions . this observed increase in viral incorporation of all major trna lys isoacceptors is in contrast to results previously obtained when overexpressing a particular trna lys isoacceptor . for example , transfection of cos7 cells with a plasmid coding for both hiv - 1 proviral dna and a trna lys3 gene results in virions with an increased concentration of trna lys3 , and a decreased concentration of trna lys1 , 2 , indicating that some trna lys packaging factor has been saturated ( huang et al ., 1994 ). based on the results presented herein , it is strongly suggested that this factor is lysrs , since increases in cytoplasmic lysrs result in the increased incorporation of all the major trna lys isoacceptors . the increase in trna lys packaging is not directly due to the increases in cytoplasmic trna lys concentrations , since overexpression of lysrs . t also results in increases in cytoplasmic trna lys concentrations , but no increase in viral incorporation of trna lys is observed . this is probably because lysrs . t does not bind to trna lys as well as lysrs . f ( fig1 ). it has been shown that removal of n - terminal sequence from yeast asprs weakens the in vitro binding of the enzyme to the trna asp , as shown by an increase in both the kd for trna binding and k m of the aminoacylation reaction of approximately 2 orders of magnitude ( frugier et al ., 2000 ). lysrs and asprs are both class iib synthetases , i . e ., they are structurally similar . however , it has been reported that human lysrs missing the n - terminal 65 amino acids did not display significantly reduced in vitro aminoacylation kinetics using an in vitro synthesized trna lys3 transcript ( shiba et al ., 1997 ). this discrepency with our band shift observations might be due either to differences in interaction using natural trna lys3 vs an unmodified trna lys3 transcript , or to the much higher concentrations of trna lys3 used in the in vitro aminoacylation reaction ( 50 - 800 fold higher than used in the band shift experiments reported here ), which might mask reduced affinities . the increase in cytoplasmic trna lys caused by overexpression of wild type or mutant lysrs could be due to several factors , including increased trna lys expression ( ie , increased transcription or nuclear export ) and / or increased trna lys stability . in yeast , uncharged trna lys acts thru a signal transduction pathway to activate the synthesis of more lysrs through increased transcription of the lysrs gene ( lanker et al ., 1992 ). presumably , this will maintain the optimum lysrs / trna lys ratio to keep all trna lys in a charged state . one could therefore predict that the cell might have a converse mechanism in which excess lysrs stimulates the synthesis of more trna lys to maintain the lysrs / trna lys ratio . however , because the increases in cytoplasmic trna lys concentration is induced by expression of mutant lysrs . t , which cannot enter the nucleus and does not bind well to trna lys , lysrs probably does not act directly on trna lys or its gene , but may instead bind another cellular factor which can alter either trna lys expression or stability . nevertheless , nuclear localization of lysrs must be necessary to fulfill some function other than directly modulating trna lys gene expression . aminoacyl trna synthetases ( aarss ) have been found to be present in the nucleus ( hopper et al ., 1998 ; lund et al ., 1998 ; sarkar et al ., 1999 ), and have been found there as high molecular weight aars complexes ( nathanson et al ., 2000 ). various functions for nuclear aarss have been proposed , including producing a more efficient export of aminoacylated trna from the nucleus ( lund et al ., 1998 ; sarkar et al ., 1999 ) which may be part of a trna proof - reading mechanism , and the regulation of rrna biogenesis in nucleoli ( ko et al ., 2001 ). nuclear localization signals ( nls ) in aarss have been predicted ( schimmel et al ., 1999 ), and our data suggests that lysrs may have an nls within the first n - terminal 65 amino acids , since removal of this segment results in the loss of ability to migrate to the nucleus ( fig1 ). the inability of lysrs . t to package trna lys is not to be confused with the normal presence of truncated lysrs in hiv - 1 . the presence of the intermediate sized lysrs fragment in virions has been correlated with trna lys incorporation into the viruses ( example 2 ). the modifications which produce the intermediate lysrs species have not yet been fully characterized , and might occur after viral packaging , i . e ., not be related to packaging the trna lys per se , but rather be required to facilitate the annealing of primer trna lys3 to the viral genome ( e . g . releasing trna lys3 so that it can interact with the retroviral genome ). furthermore , preliminary evidence using n - and c - terminal epitope tagging indicates that both c and n termini sequences are missing from the intermediate lysrs species ( data not shown ), i . e ., the naturally - occurring viral intermediate fragment is not lysrs . t . all detectable trna lys in the cell is aminoacylated ( huang et al ., 1996 ), and it is assumed that almost all trna lys is associated with lysrs . although our data indicate that lysrs is the limiting factor for trna lys viral incorporation , additional factors other than the total amount of lysrs in the cell may be involved . for example , a particular state of lysrs may be required for facilitating its interaction with gag . in the mammalian cell , lysrs is part of a high molecular weight aminoacyl trna synthetase complex ( hmw aars complex ), which in addition to containing at least 8 other aarss , contains 3 non - synthetase proteins ( mirande et al ., 1991 ). one of these , p38 , appears to act as a scaffold for assembling the aarss , and lysrs is believed to bind first , and most tightly , to p38 , and facilitate interaction with other components of the complex ( robinson et al ., 2000 ). since some of the components of this complex have already been found to be absent from hiv - 1 ( ilers and prors ( example 2 ), the question remains whether lysrs in the hmw aars complex interacts with viral protein before or after release from the complex , or if instead , some lysrs which was not part of this hmw aars complex is the source used for viral packaging . the incorporation of lysrs . t in the virion does not contradict that hmw aars is the source of the enzyme since lysrs does not require the n terminus to interact with the hmw aars ( robinson et al ., 2000 ). the cellular site of aminoacylation of the trna by aarss has also not been determined , and might occur away from the complex , with the complex acting primarily as an aars storage device . overexpressed lysrs in the cell might result in the formation of a low molecular weight lysrs / trna lys complex which can interact with gag . however , since lysrs . t is also packaged into the virions , interaction with gag probably does not require the presence of trna lys . correlation between trna lys3 aminoacylation and its incorporation into hiv - 1 the recognition and binding of aminoacyl trna synthetases ( aarss ) with their cognate trnas involves binding to the acceptor and / or anticodon arms of the trnas ( frugier et al ., 2000 ; schimmel et al ., 1987 ). for human lysrs , sequences within the anticodon arm of trna lys3 appear to play a more important role in binding lysrs than elements in the acceptor arm ( stello et al ., 1999 ). previous work has indicated that the anticodon sequence was not important for trna lys packaging into virions , i . e ., not only do trna lys3 ( anticodon suu , where s = mcm5s2 u ) and trna lys1 , 2 ( anticodon cuu ) appear to be packaged with equal efficiency , but we have reported that a mutant trna lys3 with the anticodon cua is also packaged efficiently ( huang et al ., 1996 ). however , reports have indicated a relative insensitivity of in vitro trna lys aminoacylation to mutagenesis of anticodon nucleotides u34 and u36 , compared to mutagenesis at u35 , in both an in vitro e . coli system ( tamura et al ., 1992 ) and an in vitro system using human lysrs and modified or unmodified human trna lys3 ( stello et al ., 1999 ). this agrees with previous findings that the trna lys3 with the mutant anticodon cua is still aminoacylated in vitro to 40 % wild type levels ( huang et al ., 1996 ). herein , we have constructed different trna lys3 genes mutated in the anticodon region , and expressed these genes in cos7 cells also transfected with hiv - 1 proviral dna in order to assess their incorporation into hiv and hence their modulations of trna lys3 priming processes . all mutant trna lys3 molecules contain the mutation u35g , either alone or in combination with either the u34c or u36a mutations . we show that mutations in the trna lys3 anticodon can strongly inhibit the interaction of lysrs with trna lys3 , as manifest by the inhibition of aminoacylation in vivo . the order of decreasing aminoacylation for trna lys3 anticodon mutants is : wild type uuu ( 100 %)& gt ; ugu ( 49 %)& gt ; cgu ( 40 %)& gt ; uga ( 0 %)= cga ( 0 %). the ability of trna lys3 to be aminoacylated in vivo is directly correlated with its ability to be incorporated into hiv - 1 . svc21 . bh10 is a simian virus 40 - based vector which contain wild - type hiv - 1 proviral dna and obtained from e . cohen , university of montreal . svc12 . bh10 lys3 uuu contains the hiv - 1 proviral dna plus a wild type trna lys3 gene . svc12 . bh10 lys3 cga , svc12 . bh10 lys3 cgu , svc12 . bh10 lys3 ugu , and svc12 . bh10 lys3 uga contain the hiv - 1 proviral dna plus a mutant trna lys3 gene where the anticodon has been changed from ttt to cga , cgt , tgt and tga respectively . mutant trna lys3 genes were created by pcr mutagenisis ( huang et al ., 1994 ). the amplified products were cloned into the hpa - i site of svc21 . bh10 , which is upstream of the hiv - 1 proviral dna sequence . mutations were confirmed by dna sequencing . transfection of cos7 cells with the above plasmids by the calcium phosphate method was as previously described ( mak et al ., 1997 ). viruses were isolated from cos7 cell culture medium 63 h posttransfection , or from the cell culture medium of infected cell lines . the virus - containing medium was first centrifuged in a beckman gs - 6r rotor at 3 , 000 rpm for 30 minutes and the supernatant was then filtered through a 0 . 2 μm filter . the viruses in the filtrate were then pelleted by centrifugation in a beckman ti45 rotor at 35 , 000 rpm for 1 h . the viral pellet was then purified by centrifugation with a beckman sw41 rotor at 26 , 500 rpm for 1 h through 15 % sucrose onto a 65 % sucrose cushion . total cellular or viral rna was extracted from cell or viral pellets by the guanidinium isothiocyanate procedure ( dufour et al ., 1999 ), and dissolved in 5 mm tris buffer , ph 7 . 5 . hybridization to dot blots of cellular or viral rna were hybridized with dna probes complementary to trna lys3 and trna lys1 , 2 ( khorchid et al ., 2000 ), genomic rna ( example 2 ), and ∃ actin mrna ( dna probe from ambion ). 2d page of 32pcp - 3 ′ end labeled viral rna was carried out as previously described ( khorchid et al ., 2000 ). measurement of wild type and mutant trna lys3 using rna - dna hybridization to measure the amount of trna lys3 ( wild type and mutant ) present in cellular or viral rna , we have synthesized an 18 - mer dna oligonucleotide complimentary to the 3 ′ 18 nucleotides of trna lys3 -( 5 ′ tggcgcccgmcagggac 3 ′). this probe has previously been shown to hybridize specifically with trna lys3 ( khorchid et al ., 2000 ), and was hybridized to dot blots on hybond n ( amersham ) containing known amounts of purified in vitro transcript of trna lys3 and either cellular trna or viral rna produced in cells transfected with either svc21 . bh10 alone , or svc21 . bh10 containing a wild type or mutant trna lys3 gene . the dna oligomer was first 5 ′- end labeled using t4 polynucleotide kinase and gamma - 32 p - atp ( 3000 ci / mmol , dupont canada ), and specific activities 10 8 to 10 9 cpm / ug were generally reached . approximately 10 7 cpm of oligomer was generally used per blot in hybridization reactions . for detection of specific wild type or mutant trna lys3 , dna probes complementary to the anticodon arm were used ( see fig7 ): wild type trna lys3 uuu , ( 5 ′ ccctcagattaaaagtctgatgc3 ′); trna lys3 cga , ( 5 ′ ccctcagatttcgagtctgatgc - 3 ′); trna lys3 cgu , ( 5 ′ ccctcagattacgagtctgatgc - 3 ′); trna lys3 ucu , ( 5 ′ ccctcagattacmgtctgatgc - 3 ′); and trna lys3 uca , ( 5 ′ ccctcagatttcaagtctgatgc - 3 ′). in order to specifically detect the presence of trna lys3 mutants in rna samples , blots were hybridized with 32 p labelled anticodon probes to the trna lys3 mutants in the presence of 8 - 25 fold excess of non - radioactive oligonucleotide complementary to the wild type trna lys3 anticodon arm . in vivo aminoacylation of trna lys was measured using techniques previously described ( ho et al ., 1986 ; huang et al ., 1994 ; and varshney et al ., 1991 ). to measure the extent of in vivo aminoacylation of trna lys3 , the isolation of cellular or viral rna was performed at low ph conditions required for stabilizing the aminoacyl - trna bond . the guanadinium thiocyanate procedure for isolating rna [ 5 ] was modified by including 0 . 2m sodium acetate , ph 4 . 0 in solution d , and the phenol used was equilibrated in 0 . 1m sodium acetate , ph 5 . 0 . the final isopropanol - precipitated rna pellet was dissolved in 10 mm sodium acetate , ph 5 . 0 , and stored at − 70ec until electrophoretic analysis . rna was mixed with one volume loading buffer ( 0 . 1 m sodium acetate , ph 5 . 0 , 8 m urea , 0 . 05 % bromphenol blue , and 0 . 05 % xylene cyanol ), and electrophoresed in a 0 . 5 mm thick polyacrylamide gel containing 8 m urea in 0 . 1 m sodium acetate , ph 5 . 0 . the running buffer was 0 . 1 m sodium acetate , ph 5 . 0 , and electrophoresis was carried out at 300 v , at 4 ° c ., for 15 - 18 hours in a hoefer se620 electrophoretic apparatus . rna was electroblotted onto a hybond n filterpaper ( amersham ) using an electrophoretic transfer cell ( bio - rad ) at 750 ma for 15 min , using 1 × tbe . hybridization of the blots with probes for wild type and mutant trna lys3 were performed as described above . deacylated trna was produced by treating the rna sample with 0 . 1 m tris - hcl , ph 9 . 0 at 37 ° c . for 3 hours to hydrolyze the aminoacyl linkage and provide an uncharged electrophoretic marker . western blot analysis was performed using 300 μg of cytoplasmic or nuclear proteins , as determined by the bradford assay ( barat et al ., 1989 ). cytoplasmic and nuclear extracts were resolved by sds - page followed by blotting onto nitrocellulose membranes ( gelman sciences ). detection of protein on the western blot utilized monoclonal antibodies ( anti yy1 ). western blots were analyzed by enhanced chemiluminescence ( ecl kit , amersham life sciences ) anti - mouse ( amersham life sciences ) as a secondary antibody . the sizes of the detected protein bands were estimated using pre - stained high molecular mass protein markers ( gibco / brl ). the cytoplasmic supernatant and nuclear extract were prepared from the cos7 cells as described previously ( mak et al ., 1997 ). western blot analysis was performed as above using anti - yy1 ( santa cruz ). expression of wild type and mutant trna lys3 and their incorporation into virions we have determined whether a correlation exists between the ability of a trna to be aminoacylated in vivo and to be incorporated into hiv - 1 . cos7 cells were transfected with a plasmid containing both hiv - 1 proviral dna and a wild type or mutant trna lys3 gene . as shown previously , this results in more trna lys3 being synthesized in the cytoplasm and being packaged into the viruses ( huang et al ., 1994 ). the ability of trna lys to be aminoacylated in vitro was shown to be most sensitive to sequences in the anticodon , and in particular , to u35 ( stello et al ., 1999 ). therefore , the different mutant trna lys3 being expressed all contained a u35g transition , in addition to other possible anticodon mutations ( u34c or u36a — see fig1 ). [ 0265 ] fig1 a shows dot blots of cellular or viral rna hybridized with a radioactive 18 nucleotide dna oligomer complementary to the 3 terminal 18 nucleotides of trna lys3 . the top panel represents increasing amounts of synthetic trna lys3 , and the hybridization results are plotted as a standard curve in fig1 c . the bottom 2 panels in fig1 a show dot blots of rna isolated from either cell lysate containing equal amount of b actin ( cell ) or viral lysates containing equal amounts of viral genomic rna ( viral ). western blots for determining 3 actin amounts , and dot blots for determining genomic rna amounts , are not shown . the relative total trna lys3 /∃ actin ratios are plotted in fig1 b , normalized to the value obtained in cos7 cells transfected with hiv - 1 proviral dna alone ( bh 10 ). transfection with the wild type trna lys3 gene or the mutant trna lys3 genes results in an approximate two fold increase in the cytoplasmic concentration of total trna lys3 . however , as shown in fig1 d , these cytoplasmic increases in trna lys3 did not all result in increases in trna lys3 incorporation into virions . the maximum increase in trna lys3 incorporation into virions occurred with excess wild type trna lys3 uuu ( 1 . 85 ). trna lys3 ugu and trna lys3 cgu increased 1 . 4 and 1 . 3 , respectively . trna lys3 cga showed no increase in trna lys3 incorporation , and trna lys3 uga actually showed a small decrease in packaging compared to wild type trna lys3 uuu . the experiments in fig1 measure total trna lys3 in the cytoplasm and in the virion . we have also used anticodon hybridization probes specific for each type of trna lys3 to examine their expression in the cytoplasm and incorporation into virions . this is shown in fig1 . the dot blots in panel a , which measure the amount of a specific trna lys3 present in cell or viral lysate , use rna from cell or viral lysates containing equal amounts of 3 actin or genomic rna , respectively . for each type of rna , a standard hybridization curve is generated using synthetic mutant trna lys3 transcripts . fig1 a shows the amount of trna lys3 in cytoplasm and in viruses in cells transfected with hiv - 1 alone ( bh10 ) or transfected with hiv - 1 and a trna lys3 gene ( bh10 lys3 ). fig1 b shows the amount of trna lys3 in cytoplasm and viruses for cells transfected with hiv - 1 and a mutant trna lys3 gene . in fig1 b , the wild type trna lys3 transcript was used as a control for specific hybridization of the anticodon probes . the standard curves for each type of trna lys3 are used to calculate ngms present in cell lysate or virus , and thereby taking into account any differences in efficiencies of hybridization which the different anticodon probes might have . the relative total trna lys3 /∃ actin ratios are plotted in fig1 c , normalized to the value found for cells transfected with hiv - 1 alone ( bh10 ). the results are very similar to that shown in fig1 using a dna hybridization probe which measures total trna lys3 ( wild type and mutant ). in this preparation , wild type trna lys3 is increased significantly when cells are transfected with a wild type trna lys3 gene , although not quit as much as shown in the preparation in fig1 . expression of each mutant trna lys3 in the cytoplasm are similar , and would result in an approximate 2 fold increase in total trna lys3 ( endogenous wild type and mutant ), which was shown in fig1 . the trna lys3 / genomic rna ratios in virions are shown in fig1 d , normalized to the value found for cells transfected with hiv - 1 alone ( bh10 ), and also match with similar results for total viral trna lys 3 shown in fig1 . wild type trna lys3 uuu incorporation into virions increased the ratio to 1 . 87 , indicating a relative incorporation of exogenous trna lys3 compared to endogenous trna lys3 of 0 . 87 . the relative incorporation of trna lys3 ugu and trna lys3 cgu was 0 . 50 and 0 . 37 , respectively , while trna lys3 cga and trna lys3 uga showed relative incorporations of 0 . 013 and 0 . 29 . these data indicate that wild type or mutant trna lys3 are expressed at approximately equal levels in the total cell lysate , but some mutant trnas are not incorporated into virions as well as others . one possible explanation could be that some mutant trnas are not exported with equal efficiency out of the nucleus . to test this we lysed cells , and separated nuclei from cytoplasm by low speed centrifugation . dot blots of the rna in cytoplasmic fraction , representing equal amouts of b actin , were hybridized with either the 3 ′ terminal dna probe , which hybridizes to all trna lys3 s ( fig1 a ) or with anticodon probes specific for each trna lys3 ( fig1 b - e ). in panels b - e , rna from the bh10 cytoplasmic fraction was used as the control to show hybridization specificity for each anticodon probe . it can be seen that , as concluded in fig1 which used total cell lysates , that all trna lys3 s are expressed approximately equally . panel f at the bottom of the figure demonstrates the efficiency of the separation of nuclear and cytoplasmic fractions , ie , the nuclear transcription factor yyi , which concentrates in the nucleus , is only detected in that fraction . aminoacylation of wild type and mutant trna lys3 in vivo the aminoacylation state of the wild type and mutant cellular trna lys3 were examined . the electrophoretic mobility of acylated trna in acid - urea page has been reported to be slower than the deacylated form , and this property can be used to determine the degree of trna aminoacylation ( huang et al ., 1996 ). fig1 shows northern blots of cellular and viral rna samples electrophoresed in acid - urea gels , blotted onto hybond nl filterpaper , and hybridized with radioactive trna lys3 dna probes . in panel a , cellular trna was hybridized with the 18 nucleotide dna oligomer complementary to the 3 ′ 18 nucleotide terminus of trna lys3 , while in panels b - e , the cellular trna was hybridized with the anticodon probes specific for different mutant trnas . lane 1 in panel a represents wild type trna lys3 deacylated in vitro to mark where deacylated trna lys3 migrates in the gel . as has previously been reported ( huang et al ., 1996 ), in cells transfected with either the wild type trna lys3 gene ( lane 2 ), or not transfected with any trna lys3 gene ( lane 3 ), the trna detected is entirely in the aminoacylated form . this is shown graphically in panel f , where cytoplasmic aminoacylation is given as 100 %. it can also be seen in panel a that a majority of the total trna lys3 is aminoacylated in cells transfected with genes coding for trna lys3 cgu ( lane 5 ) and trna lys3 ugu ( lane 6 ), with a larger proportion of total trna lys3 being in the deacylated form in cells transfected with genes coding for trna lys3 cga ( lane 4 ) and trna lys3 uga ( lane 7 ). since total trna lys3 consists of both endogenous trna lys3 uuu and exogenous mutant trna lys3 , the data in panel a gives us an indirect view of the ability of the mutant trna lys3 s to be aminoacylated . we therefore used anticodon dna probes specific for the different mutant trna lys3 s ( panels b - e ). lanes 8 , 11 , 14 , and 17 represent the corresponding mutant trna lys3 samples which have been deacylated in vitro , while lanes 10 , 13 , 16 , and 19 contain cellular rna from cells transfected only with hiv - 1 proviral dna , and show the hybridization specificity of the anticodon probes . it is clear from the data in these panels that trna lys3 ugu ( lane 9 ) and trna lys3 cgu ( lane 12 ) are aminoacylated better than trna lys3 cga ( lane 15 ) and trna lys3 uga ( lane 18 ). the percentage of each mutant trna lys3 which is aminoacylated is also shown graphically in panel f . herein , we have shown that the ability of trna lys3 to be incorporated into hiv - 1 is closely correlated with its ability to be aminoacylated . aminoacylation is dependent upon the binding of lysrs to trna lys3 , demonstrating that this interaction is required for trna lys incorporation into virions . whether aminoacylation itself is required for viral trna lys packaging cannot be inferred from this data . other data presented herein is consistent with lysrs binding to trn lys playing an important role in trna lys packaging , however . for example , when cos7 cells are cotransfected with plasmids containing both hiv - 1 proviral dna and a lysrs gene , the viral trna lys concentration goes up 2 fold . on the other hand , transfection with a mutant , n - terminally truncated lysrs gene , which produces lysrs unable to bind to trna lys , does not result in any increase in trna lys packaging , although the mutant lysrs is still packaged into the virion ( example 2 ). the data presented in this work supports a model in which the trna lys3 / lysrs interaction is important for trna lys3 incorporation into viruses . however , the anticodon sequence has also been implicated in the in vitro binding of mature reverse transcriptase to either purified trna lys3 ( sarih - cottin et al ., 1992 ) or trna lys3 transcripts ( barat et al ., 1989 ; wohrl et al ., 1993 ). since rt sequences in gagpol have been implicated in an interaction with trna lys3 during it incorporation into virions ( khorchid et al ., 2000 ; mak et al ., 1994 ), mutant anticodon might also weaken this trna lys3 / gag pol interaction . both in vitro studies ( dufour et al ., 1999 ; mishima et al ., 1995 ) and in vivo studies ( khorchid et al ., 2000 ) indicate that the thumb domain sequences within rt probably interact with trna lys3 . in vitro cross linking studies indicate an interaction between rt peptides containing the thumb domain and either synthetic ( mishima et al ., 1995 ) or purified ( dufour et al ., 1999 ) trna lys3 . in vivo , it has been shown that trna lys3 incorporation into hiv - 1 is not affected by deletion of the in domain in pr160 gag - pol , nor by further deletion of the rnaseh and connection subdomains within the rt domain of pr160 gag - pol . however , trna lys3 packaging is severely inhibited by further deletions into the thumb subdomain ( khorchid et al ., 2000 ). however , the site of rt interaction on the trna lys3 is in question . while one report indicates an interaction in vitro between the thumb domain and the trna lys3 anticodon loop ( mishima et al ., 1995 ), another report indicates an interaction in vitro between the thumb domain and the 3 ′ terminus of trna lys3 ( dufour et al ., 1999 ). these differences may be due to the use of synthetic trna lys3 in the former case , and purified trna lys3 in the latter case . using mutational analysis , arts et al ., ( arts et al ., 1998 ) also found evidence for an in vitro interaction between the anticodon loop of trna lys3 and a small crevice in the p66 thumb domain of rt . herein , mutations in certain amino acids in the thumb subdomain ( k249 , r307 ) were found to inhibit the interaction of mature rt with the trna lys3 anticodon domain in vitro . however , since these same rt amino acid mutations had no effect upon trna lys3 packaging in vivo ( khorchid et al ., 2000 ), there is no evidence for an in vivo interaction between rt sequences in gagpol and the trna lys3 anticodon during trna lys3 packaging . in summary , the present invention shows a positive correlation between the amount of trna lys3 incorporated into virions , the amount of trna lys3 annealed to the viral genome , and the infectivity of hiv virions . furthermore , the trna lys - binding protein , lysyl - trna synthetase ( lysrs ), is selectively packaged along with trna lys into hiv - 1 , and the amount of lysrs in the virus determines the amount of trna lys packaged into the virus . in addition , the ability of trna lys3 to be incorporated into hiv - 1 or to affect lysrs - facilitated processes associated with trna lys3 priming of rt , is shown to be closely correlated with its ability to be aminoacylated , and hence of its binding to lysrs . the viral precursor protein pr55 gag alone will package lysrs into pr55 gag particles , independently of trna lys . with the additional presence of the viral precursor protein pr160 gag - pol , trna lys and lysrs are both packaged into the particle . while the predominant cytoplasmic lysrs has an apparent mr = 70 , 000 , viral lysrs associated with trna lys packaging is truncated and has an apparent mr = 63 , 000 . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims . berkowitz et al ., 1996 , rna packaging , p . 177 - 218 . in h . g . krausslich ( ed . ), morphogenesis and maturation of retroviruses ., vol . 214 . springer - verlag , berlin heidelberg n . y . leis et al ., 1993 . regulation of initation of reverse transcription of retroviruses , p . 33 - 47 . in a . m . skalka , and s . p . goff ( eds ), reverse transcriptase , vol . 1 . cold spring harbor laboratory press , new york , n . y . meinnel et al ., 1995 , aminoacyl - trna synthetases : occurrence , structure , and function ., p . 251 - 292 . in d . soll , and u . l . rajbhandary ( eds ), trna : structure , biosynthesis , and function . asm press , washington , d . c . mirande m ., 1991 , progress in nuclei acid research and molecular biology 40 : 95 - 142 . swanstrom et al ., 1997 , synthesis , assembly , and processing of viral proteins ., p . 263 - 334 . in j . coffin , s . hughes , and h . varmus ( eds ), retroviruses . cold spring harbor laboratory press , plainview , n . y . waters et al ., 1977 , prog . nucleic acid res . mol . biol . 20 : 131 - 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