bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Chemical Modification and Fragmentation on Antigenic Determinants of Internal Protein p30 and Surface Glycoprotein gp70 of Type C Retroviruses
Authors: Versteegen Rosemary J. , Oroszlan Stephen
Published: January 2020

The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of α and ε amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH
2
-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27.
Glycopeptides of murine leukemia viruses. II. Comparison of xenotropic and dual-tropic viruses
Authors: Kemp M C , Famulari N G , O'Donnell P V , Compans R W
Published: January 2020

The glycosylation patterns of the gp70 glycoproteins of xenotropic and dualtropic murine leukemia virus (MuLV) were compared with those of ecotropic viruses. Ecotropic viruses contain a large glycopeptide size class designated G1 (molecular weight, approximately 5100), and such glycopeptides were not detected in xenotropic viruses grown in mink cells nor in dual-tropic viruses grown in mouse or mink lung cells. Both xenotropic and dual-tropic MuLV had glycopeptide size classes designated G2, G3, and G4 (molecular weights, approximately 2900, 2,200, and 1,500, respectively). G2 glycopeptides of xenotropic and dual-tropic MuLV were shown to be resistant to endo-beta-N-acetylglucosaminidase H, whereas G3 and G4 glycopeptides were susceptible. The relative abudance of glycopeptide G3 was increased in xenotropic and dual-tropic viruses as compared with ecotropic viruses, whereas the relative amount of G4 was decreased in xenotropic viruses. The similarity in the glycosylation patterns of a number of xenotropic and dual-tropic viruses suggests that glycosylation sites are highly conserved within the env gene products of each of these classes of viruses.
Structure and Functions of the Bacteriophage P22 Tail Protein
Authors: Berget Peter B. , Poteete Anthony R.
Published: January 2020

The product of gene
9
(gp9) of
Salmonella typhimurium
bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an
s
20,w
of 9.3S, a
D
20,w
of 4.3 × 10
−7
cm
2
/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from
supF
cells infected with a phage strain carrying an amber mutation in gene
9.
Phage “tailed” with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.
Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration
Authors: Dorsch-Häsler Karoline , Fisher Paul B. , Weinstein I. Bernard , Ginsberg Harold S.
Published: January 2020

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5
ts
125 and H5
ts
107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C
0
t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5
ts
125 or H5
ts
107 at either the semipermissive (36°C) or nonpermissive (39.5°C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases
Xba
I,
Eco
RI, or
Hin
dIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol.
98
: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration.
Genetic Variation During Persistent Reovirus Infection: Presence of Extragenically Suppressed Temperature-Sensitive Lesions in Wild-Type Virus Isolated from Persistently Infected L Cells
Authors: Ahmed Rafi , Chakraborty P. R. , Graham A. F. , Ramig R. F. , Fields B. N.
Published: January 2020

Persistent reovirus infection of L cells was established with a serially passaged stock of temperature-sensitive (
ts
) mutant C(447) containing greater than 90% defective interfering particles. Within a month after establishment of the carrier culture, the
ts
mutant was replaced by virus that expressed the wild-type (
ts
+
) temperature phenotype (R. Ahmed and A. F. Graham, J. Virol.
23
:250-262, 1977). To determine whether the
ts
+
phenotype of the virus was due to intragenic reversion or to the presence of an extragenic mutation suppressing the original
ts
defect, several clones were backcrossed to wild-type reovirus, and the progeny of each cross were screened for temperature sensitivity. The results indicated that the original
ts
C lesion had reverted. However, in two of the seven clones examined, new
ts
lesions were found. These new
ts
lesions appeared phenotypically as
ts
+
due to the presence of extragenic suppressor mutations. Temperature-sensitive mutants representing three different groups were rescued from one suppressed clone, indicating that this
ts
+
clone contained multiple
ts
lesions. Among the
ts
mutants rescued were the initial isolates of a new recombination group which we have designated H. Some of the
ts
mutants rescued from the suppressed clones are capable of interfering with the growth of wild-type reovirus and may play a role in maintaining the carrier state. The results of this study show that persistently infected L cells contain a genetically heterogeneous population of reovirus even though all virus clones express the
ts
+
phenotype. It is thus critical to distinguish between genotype and phenotype when analyzing viruses that emerge during persistent infection.
BK virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology
Authors: Yang R C , Young A , Wu R
Published: January 2020

The DNA sequence of the early region of the human papovavirus BK (MM strain) was determined. A potential initiation signal for translation is located at nucleotides 3,047 to 3,045 or map position 0.614. Extending counterclockwise from this AUG signal there is only one open reading frame, which can code for a putative t antigen of 100 amino acids in length. If the early mRNA of BKV is spliced, then the regions between nucleotides 3,047 to 2,808 and 2,725 to 884 can code for a T antigen 694 amino acids in length. The sequences of the deduced T antigens in BK virus share 71% amino acid homology with those in simian virus 40, whereas the coding sequences of the two viruses share 70% DNA homology. Comparison of DNA sequences and evaluation of homology measurements between these two viruses are discussed.
Biochemical characterization of endogenous type C virus information in differentiated and undifferentiated murine teratocarcinoma-derived cell lines
Authors: Emanoil-Ravicovitch R , Hojman-Montes-de-Oca F , Robert J , Garcette M , Callahan R , Peries J , Boiron M
Published: January 2020

Undifferentiated teratocarcinoma cells express sixfold-higher levels of endogenous xenotropic type C virus-related RNA than differentiated cells. Three species of polyadenylated viral RNA (35S, 24S, and 14S) have been identified in the undifferentiated teratocarcinoma cells. Paradoxically, neither viral particles nor viral proteins have been detected in these cells.
Defective mutant of Sindbis virus with a smaller-molecular-weight form of the E1 glycoprotein
Authors: Leone A , Colantuoni V , Pontarelli G , Cancedda R
Published: January 2020

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.
Is cytoskeleton involved in vesicular stomatitis virus reproduction?
Authors: Genty N , Bussereau F
Published: January 2020

CER cells infected with vesicular stomatitis virus showed a morphology similar to that observed after cytochalasin B treatment. Temperature-sensitive mutants affected in envelope protein maturation did not induce those morphological changes at a nonpermissive temperature. In addition, the cytoskeleton was not implicated in vesicular stomatitis virus reproduction.
Cytoplasmic T antigens of mouse and human cells transformed by a simian virus 40 tsA mutant
Authors: Dubbs D R , Otsuka H , Kit S
Published: January 2020

Simian virus 40 T antigens accumulate in the cytoplasm of simian virus 40 tsA207 transformants of primary mouse kidney or human retinoblastoma cells grown at 40 degrees C in 10% serum.
Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins
Authors: Wolstenholme A J , Barrett T , Nichol S T , Mahy B W
Published: January 2020

Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.
Characterization of a 170,000-dalton polyprotein encoded by the McDonough strain of feline sarcoma virus
Authors: Van de Ven W J , Reynolds F H , Nalewaik R P , Stephenson J R
Published: January 2020

In this study, we demonstrated the expression of a 170,000-Mr polyprotein in each of several McDonough feline sarcoma virus (FeSV)-transformed mink cell clones and one McDonough FeSV-transformed rat clone. This polyprotein designated McDonough FeSV P170, contained feline leukemia virus (FeLV) p15, p12, and p30 immunological determinants and shared two of its five [35S]methionine-labeled tryptic peptides with FeLV Pr180gag-pol. Both of these peptides were shown to be specific to the p30 component of Pr180gag-pol. The remaining McDonough FeSV P170 methionine-containing peptides were not represented within either FeLV Pr180gag-pol or Pr82env. Of interest, of the three peptides specific to the nonstructural component of McDonough FeSV P170, one was also represented in the 115,000-Mr polyproteins encoded by the Gardner and Snyder-Theilen strains of FeSV. These findings raise the possibility that the nonstructural components of polyproteins encoded by each of the three independently derived feline transforming viruses contained both common and unique regions. Moreover, if the sequences encoding these components are involved in transformation, as appears to be the case, our findings establish that the position of their insertion within the gag-pol region of the FeLV genome can vary among individual isolates.
Three independent isolates of feline sarcoma virus code for three distinct gag-x polyproteins
Authors: Ruscetti S K , Turek L P , Sherr C J
Published: January 2020

Cells nonproductively transformed by the Snyder-Theilen, Gardner-Arnstein, and McDonough strains of feline sarcoma virus synthesize gag-x polyproteins of 78,000, 100,000, and 180,000 daltons, respectively. These feline sarcoma virus-coded products were precipitated by antisera to polypeptides encoded by the gag gene of feline leukemia virus and by rat antisera raised to feline sarcoma virus-transformed rat tumor cells. Precipitation with rat antisera absorbed with feline leukemia virus showed that the x-portions of the three gag-x proteins were each antigenically distinct, suggesting that the src genes of the three independent isolates are not identical. Anti-x sera did not precipitate products from radiolabeled cat lymphoid tumor cells (FL74) and therefore lacked reactivity to the feline leukemia virus-induced tumor-specific antigen, FOCMA.
Patterns of Transcription of Human Cytomegalovirus in Permissively Infected Cells
Authors: Demarchi Jean M. , Schmidt Carla A. , Kaplan Albert S.
Published: January 2020

The rate of accumulation of cytomegalovirus transcripts in permissively infected human embryonic lung (HEL) cells was analyzed at various times after infection by hybridization of infected cell RNA to undigested or restriction endonuclease-digested cytomegalovirus DNA fixed to nitrocellulose filters. Differences in patterns of transcript accumulation were determined by measuring the abundance levels of RNA which hybridized to different
Hin
dIII-,
Xba
I-, or
Eco
RI-generated fragments of cytomegalovirus DNA. The results showed that a small but significant amount of cytomegalovirus RNA was detectable within the first 3 h after infection and that the rate of accumulation of these transcripts was static during the first 24 h, but increased thereafter. In general, the viral transcripts accumulating in infected cells could be divided into three classes. Immediate-early RNA (synthesized in the absence of protein synthesis in infected cells) hybridizes predominantly to a very restricted part of the genome and can be identified during the first 2 to 4 h postinfection. Early RNA (synthesized up to about 24 h after infection) originates from most regions of the genome but is characterized by the presence of transcripts which hybridize in great abundance to certain fragments. Late RNA (synthesized after 24 h, i.e., after the onset of viral DNA synthesis) hybridizes in approximately equal abundance to most regions of the viral genome. These results showed that a block in the transition from immediate-early to early RNA did not account for the extended period of time that elapses between the time of infection and the initiation of viral DNA synthesis. Interestingly, despite rapid adsorption and penetration and a static level of accumulation of transcripts in the cultures during the first 24 h, the number of cells that synthesized detectable amounts of viral antigens increased steadily during this time.
Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions
Authors: Brady John N. , Lavialle Christian , Salzman Norman P.
Published: January 2020

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-
N-N′
-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP
1
and VP
2
in addition to the proteins found associated with the viral minichromosome, i.e., VP
3
and histones H
2
A, H
2
B, H
3
, and H
4
. Our results suggest that the association of VP
1
, VP
2
, or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of
Escherichia coli
RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the “early” SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP
1
or VP
2
or both enhances the transcriptional activity and suggest that these “late” viral proteins may play a role in the regulation of expression of the SV40 genome.
Partial replicas of UV-irradiated bacteriophage T4 genomes and their role in multiplicity reactivation
Authors: Rayssiguier C , Kozinski A W , Doermann A H
Published: January 2020

A physicochemical study was made of the replication and transmission of UV-irradiated T4 genomes. The data presented in this paper justify the following conclusions. (i) For both low and high multiplicity of infection there was abundant replication from UV-irradiated parental templates. It exceeded by far the efficiency predicted by the hypothesis that a single lethal hit completely prevents replication of the killed phage DNA: i.e., some dead phage particles must replicate parts of thier DNA. (ii) Replication of the UV-irradiated DNA was repetitive as shown by density reversal experiments. (iii) Newly synthesized progeny DNA originating from UV-irradiated templates appeared as significantly shorter segments of the genomes than progeny DNA produced from non-UV-irradiated templates. A good correlation existed between the number of UV hits and the number of random cuts that would be needed to reduce replication fragments to the length observed. (iv) The contribution of UV-irradiated parental DNA among progeny phage in multiplicity reactivation was disposed in shorter subunits than was the DNA from unirradiated parental phage. It is important to emphasize that it was mainly in the form of replicative hybrid. These conclusions appear to justify excluding interparental recombination as a prerequisite for multiplicity reactivation. They lead directly to some form of partial replica hypothesis for multiplicity reactivation.
Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ
Authors: Deppert W , Hanke K , Henning R
Published: January 2020

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture.
Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis
Authors: Glorioso J C , Levine M , Holland T C , Szczesiul M S
Published: January 2020

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.
Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells
Authors: Leis J P , Scheible P , Smith R E
Published: January 2020

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.
Interaction of mycoplasma virus type 2 with cellular components of Acholeplasma laidlawii strain JA1
Authors: Al-Shammari A J , Smith P F
Published: January 2020

Mycoplasma virus type 2 was shown to adsorb specifically to intact cells, membranes, and lipoglycan of Acholeplasma laidlawii strain JA1 but not to these components of Acholeplasma oculi. The oligosaccharide chain of the lipoglycan defined the specificity of the receptor site since deacylation not only did not reduce adsorption but increased it threefold. Actual adsorption of virus to lipoglycan was demonstrated by sucrose density gradient separation of the virus-lipoglycan complex. A strain of A. laidlawii, JA1r, resistant to infection with mycoplasma virus type 2, was incapable of adsorbing the virus and was devoid of lipoglycan.
Transcripts of the adeno-associated virus genome: mapping of the major RNAs
Authors: Green M R , Roeder R G
Published: January 2020

The four major adeno-associated virus type 2 (AAV2)-specific RNAs were mapped on the linear viral genome by a variety of biochemical techniques, including S1 nuclease and exonuclease VII mapping, RNA gel-transfer hybridization, and analysis of reverse transcriptase extension products. All the major AAV2 RNAs were derived from the minus DNA strand and had 3' termini at position 96. The nucleus-specific 4.3- and 3.6-kilobase (kb) RNAs had 5' termini at positions 6 and 19, respectively. The 5' terminus of the 2.6-kb RNA mapped to position 38.5. The predominant 2.3-kb AAV2 mRNA was spliced and contained a short leader sequence (approximately 50 nucleotides) which mapped to position 38.5, coincident with the 5' terminus of the 2.6-kb RNA. The 5' end of the body of the 2.3-kb RNA mapped to position 46.5. These results are discussed in terms of the involvement of single versus multiple promoters (for transcription) and RNA splicing mechanisms in the generation of the AAV2 RNAs.
Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences
Authors: Heilman C A , Law M F , Israel M A , Howley P M
Published: January 2020

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.
Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles
Authors: Peters G G , Hu J
Published: January 2020

Mutants of avian sarcoma virus which lack a functional DNA polymerase were found to be nonselective in the incorporation of host cell tRNA's into virus particles. In contrast, mutants which possess a functional DNA polymerase but lack the viral genome RNA contained a specific subset of the host cell tRNA population, indistinguishable from that of the wild-type virus. Thus the reverse transcriptase, and not the viral RNA, is probably the major factor determining which tRNA's are incorporated into avian sarcoma virus particles. Supporting evidence was obtained in an in vitro binding assay between purified reverse transcriptase and unfractionated cellular tRNA's. However, the subset of tRNA's which associated with the genome in the 70S complex was determined primarily by the viral RNA. In the absence of DNA polymerase, the 70S RNA complex in mature virus particles contained the normal complement of associated tRNA's with the exception of tRNATrp, the primer for RNA-directed DNA synthesis.
Envelope assembly mutant of rous sarcoma virus
Authors: Steimer K S , Boettiger D
Published: January 2020

The properties of a novel nonconditional envelope mutant of Rous sarcoma virus, rdPN3/2-SR-D, defective in the assembly of viral glycoproteins into mature virions, are described.
Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis
Authors: Stow N D
Published: January 2020

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules.
Nucleotide sequence at polyoma VP1 mRNA splice sites
Authors: Srivatsan E S , Deininger P L , Friedmann T
Published: January 2020

Double-stranded DNA complementary to total cytoplasmic polyadenylated RNA isolated late in infection from polyoma virus-infected mouse 3T6 cells was cloned in Escherichia coli by using the large HindIII-BamHI fragment of pBR322 plasmid DNA. Polyoma-specific DNA inserts were detected by hybridization, and then nucleotide sequences were determined from two clones. The sequence of the (formula see text) with prototypical mammalian splice sites, and the dashed arrows indicate possible alternative splice sites leading to the same spliced product. A sequence of 897 nucleotides was spliced out of the primary transcript during the processing of the mature VP1 mRNA. Restriction enzyme mapping with four other independently isolated clones indicates that these are the major splicing signals for the VP1 message. The distal splice site is 48 nucleotides upstream from the initiator codon.
Monovalent cation metabolism and cytopathic effects of poliovirus-infected HeLa cells
Authors: Nair C N
Published: January 2020

To better understand the significance of 22Na+ accumulation by poliovirus-infected HeLa cells (C. N. Nair, J. W. Stowers, and B. Singfield, J. Virol. 31:184, 1979), measurements of cellular Na+, K+, and Cl- contents, volume, and density were carried out at intervals after infection. In addition, the rates of 22Na+ washout from infected and control cells were determined. Starting at around 3 h postinfection, the Na+ content of infected cells increased, whereas the K+ content decreased progressively, resulting in a net loss in the monovalent cation content decreased progressively, resulting in a net loss in the monovalent cation content per cell. The loss in cellular chloride content exceeded that in monovalent cation content. The kinetics of 22Na+ washout from infected and control cells revealed the presence of an extra Na+ compartment in infected cells. A net loss in the monovalent cation activity of infected cells was indicated by the loss of cell water as reflected in a decrease in cell volume and an increase in cell density. In spite of a net loss in monovalent cation content per cell, Na+ accumulation coupled with cell shrinkage resulted in substantial increases in the concentrations of not only Na+ but also K+. The results suggested a possible role for tonicity change in the morphological lesions of poliovirus cytotoxicity.
Recurring defective variants of simian virus 40 containing monkey DNA segments
Authors: Papamatheakis J , Lee T N , Thayer R E , Singer M F
Published: January 2020

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.
Comparison of ribonucleotide sequences from the genome of vesicular stomatitis virus and two of its defective interfering particles
Authors: Hagen F S , Huang A S
Published: January 2020

RNA genomes from standard vesicular stomatitis virus and two defective interfering (DI) particles dI 0.33 (DI-T) and DI 0.52, were purified and digested with RNase T1. The resulting oligonucleotides were labeled at the 5' end with [32P]ATP and separated by two-dimensional electrophoresis in polyacrylamide gels. All of the major oligonucleotides containing 20 or more nucleotides were sequenced. Those oligonucleotides that were thought to be in common by their migration on polyacrylamide gels actually did have identical sequences. Those oligonucleotides thought to be unique to the DI RNAs either differed by only one nucleotide from oligonucleotides of the standard RNA or contained new sequences which were complementary to known sequences at the 5' end. These data indicate that RNAs from DI particles are not simple deletions but contain point mutations and additional complementary sequences.
Replicating, differentiated macrophages can serve as in vitro targets for transformation by avian myeloblastosis virus
Authors: Durban E M , Boettiger D
Published: January 2020

Pure cultures of chicken macrophages were characterized functionally and transformed by avian myeloblastosis virus. Transformed cells exhibited an altered function. The efficiency of transformation was limited by the mitotic activity of the macrophages.
Simian virus 40 early mRNA's in lytically infected and transformed cells contain six 5'-terminal caps
Authors: Kahana C , Gidoni D , Canaani D , Groner Y
Published: January 2020

Late simian virus 40 (SV40) mRNA contains eight different cap structures which we have previously identified and mapped on the viral genome. As reported here, 5'-cap heterogeneity is a common feature to both the early and the late SV40 mRNA's. methyl-3H-labeled viral mRNA was purified from cells infected at 41 degrees C with SV40 mutant tsA209. Three different cap cores were identified: m7GpppGm, m7GpppCm, and m7GpppAm. An average of three to four m6A residues per mRNA molecule was found. RNase T2-resistant 32P-labeled early caps from tsA209-infected cells isolated and characterized. Six distinct cap I structures were identified: m7GpppCmpU (30%), m7GpppGmpC (24%), m7GpppAmpG (18%), m7GpppGmpU (13%), m7GpppGmpG (12%), and m7GpppAmpU (3%). A similar 5'-end heterogeneity was observed in early SV40 mRNA from BSC-1 cells infected with wild-type SV40 strain 777 in the presence of cytosine arabinoside and in the SV40 UV-transformed permissive line C-6. Five of these capped dinucleotides are complementary to DNA sequences at 0.66 map unit in a region previously identified by the primer extension method (Reddy et al., J. Virol. 30:279-296, 1979; Thompson et al., J. Virol. 31:437-438, 1979) as the 5' end of the early message. DNA sequences upstream from this region contain the TATTTAT (Hogness-Goldberg box), which is missing from upstream of the 5'-cap sites of late SV40 mRNA. Thus, 5'-end heterogeneity is not necessarily related to the presence or the absence of this putative transcriptional "initiation signal." When the possibility that SV40 5' caps represent transcriptional initiation sites is considered, the data also suggest that, on SV40 DNA, eucaryotic RNA polymerase II initiates transcription at multiple nucleotide sequences, including pyrimidines.
Detection and characterization of multiple forms of simian virus 40 large T antigen
Authors: Fanning E , Nowak B , Burger C
Published: January 2020

Subclasses of simian virus 40 large T antigen in simian virus 40-transformed and -infected cells separated by zone velocity sedimentation in sucrose density gradients have been characterized. Three forms of large T antigen were distinguished: a 5 to 6S form, a 14 to 16S form, and a 23 to 25S form. These forms appeared to differ biochemically and biologically. Differential labeling experiments suggested that the 5 to 6S form was less highly phosphorylated than the faster-sedimenting forms. The 23 to 25S form which was complexed with one or more host phosphoproteins, as reported recently (D. P. Lane and L. V. Crawford Nature [London] 268:261-263, 1979; F. McCormick and E. Harlow, J. Virol. 34: 213-224, 1980), was prominent in extracts of transformed cells, but was also detected in productively infected cells. Pulse-chase experiments suggested that the 5 to 6S large T antigen is a precursor of the more stable, faster-sedimenting forms of T antigen. Monkey cells infected with a tsA mutant of simian virus 40 at 41 degrees C contained only 5 to 6S large T antigen, implying that this form is not active in the initiation of simian virus 40 DNA replication. In pulse-chase, shift-down experiments, DNA replication resumed, and the 5 to 6S large T antigen which had accumulated at 41 degrees C was partially converted at 33 degrees C to a fast-sedimenting form. However, shift-up experiments demonstrated that the fast-sedimenting large T antigen, once formed, remained stable at 41 degrees C, although it was unable to function in initiation. These experiments suggest that different biological functions of large T antigen may be carried out by different subclasses of this protein.
Protein kinase activity immunoprecipitated from adenovirus infected cells by sera from tumor-bearing hamsters
Authors: Branton P E , Lassam N J , Downey J F , Yee S P , Graham F L , Mak S , Bayley S T
Published: January 2020

Earlier, we reported (N. J. Lassam, S. T. Bayley, F. L. Graham, and P. E. Branton, Nature (London) 277:241-243, 1979) detecting protein kinase activity when cytoplasmic extracts of human adenovirus type 5 (Ad5)-infected KB cells immunoprecipitated with 14b antitumor serum directed against the transforming proteins of Ad5, were incubated with [gamma-32P]ATP. Here we show that in the in vitro assay this kinase phosphorylated both the heavy chain of immunoglobulin G and polypeptide than comigrated on sodium dodecyl sulfate gels with the 58,000-dalton Ad5 antigen. It also phosphorylated added histone H3. Evidence is presented that the protein kinase activity found with extracts from Ad5-infected cells is not due to nonspecific trapping of cellular enzymes in immune complexes, but to an enzyme which is distinct from kinases detected at background levels in controls. Serine and threonine were the major phosphorylated amino acids, and essentially no phosphotyrosine was detected. Protein kinase activity detected in Ad12-infected cells immunoprecipitated by an antiserum derived from hamsters bearing Ad12-induced tumors appeared to be immunologically distinct from that immunoprecipitated from Ad5-infected cells by 14b serum.
Revised transcription map of the late region of bacteriophage T7 DNA
Authors: Carter A D , Morris C E , McAllister W T
Published: January 2020

The genes in the late region of the bacteriophage T7 genome are transcribed by a phage-specified RNA polymerase in two temporal classes (II and III). Through the use of an agarose-formaldehyde gel system that permits the resolution of high-molecular-weight RNAs, transcription of template DNA that had been cut with various restriction endonucleases, and transcription of isolated restriction fragments from the class II region, six previously unreported promoters utilized by the phage RNA polymerase in vitro have been mapped between 28 and 46% of the T7 genome. No evidence could be found for the existence of a promoter at 61% that had previously been reported to give rise to a diffuse band (species I RNA) in acrylamide-agarose gels.
Properties of simian virus 40 small t antigen overproduced in bacteria
Authors: Thummel C S , Burgess T L , Tjian R
Published: January 2020

We constructed a series of bacterial plasmids which contained the Escherichia coli lac promoter fused to a simian virus 40 restriction fragment coding for small t antigen. These plasmids expressed different levels of intact viral protein depending on the length of the constructed ribosome binding site. Small t antigen synthesized by the most efficient producer, HP1, constituted 0.5 to 1% of the total cellular protein. On the basis of extensive characterization by immunoprecipitation, gel electrophoresis, isoelectric focusing, tryptic fingerprint analysis, and chromatographic properties, this plasmid-encoded protein was virtually identical to authentic simian virus 40 small t antigen. Partial purification of the HP1-encoded and authentic small t antigens revealed the presence of both monomeric and multimeric forms.