bluuebunny/crossref_metadata_2025 huggingface dataset:
Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum
Authors: Kabcenell A K , Poruchynsky M S , Bellamy A R , Greenberg H B , Atkinson P H
Published: January 2020

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.
Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells
Authors: Murphy C I , Weiner B , Bikel I , Piwnica-Worms H , Bradley M K , Livingston D M
Published: January 2020

The insect baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector for the simian virus 40 (SV40) small t (t) and large T (T) antigens. Spodoptera frugiperda (SF9) cells infected with recombinant viruses encoding these proteins produced approximately 1 to 2 micrograms of t and up to 30 micrograms of T per 3 X 10(6) cells. The former was highly soluble after Nonidet P-40 extraction of the infected cells, unlike its Escherichia coli-produced counterpart. Both SF9-produced proteins were of authentic size and could be readily immunoprecipitated by specific antibodies. Single-step immunoaffinity chromatography was used to purify the two proteins to near homogeneity, with yields averaging 70% in each case. Experiments to test the biological activity of the baculovirus SV40 proteins showed that SF9 t was capable of associating with two of the cellular proteins reported to bind to t in SV40-infected mammalian cells. Moreover, SF9 T had ATPase activity comparable to that of T produced in monkey cells, exhibited helicase activity and SV40 origin-specific DNA binding, and was active in the SV40 DNA replication assay in vitro. Thus, the SV40 T antigens produced in insect cells can be used in future studies of their biochemical roles in vitro and in vivo.
Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus
Authors: Lillehoj E P , Salazar F H , Mervis R J , Raum M G , Chan H W , Ahmad N , Venkatesan S
Published: January 2020

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.
Binding of cellular proteins to the regulatory region of BK virus DNA
Authors: Markowitz R B , Dynan W S
Published: January 2020

The human papovavirus BK has a noncoding regulatory region located between the divergently transcribed early and late coding regions. Many strains of BK virus (BKV) have direct DNA sequence repeats in the regulatory region, although the number and extent of these repeats varies widely between independent isolates. Until recently, little was known about the individual functional elements within the BKV regulatory region, and the biological significance of the variable repeat structure has been unclear. To characterize the interaction between sequences in the BKV regulatory region and host cell transcription factors, we have carried out DNase I footprinting and competitive binding experiments on three strains of BKV, including one strain that does not contain direct sequence repeats. We have used relatively crude fractions from HeLa cell nuclear extracts, as well as DNA affinity-purified preparations of proteins. Our results demonstrate that BK(Dunlop), BK(WW), and BK(MM) each contain multiple binding sites for a factor, NF-BK, that is a member of the nuclear factor 1 family of transcription factors. We predict the presence of three to eight binding sites for NF-BK in the other strains of BKV for which a DNA sequence is available. This suggests that the binding of this protein is likely to be required for biological activity of the virus. In addition to NF-BK sites, BK(WW) and BK(MM) each contain a single binding site for transcription factor Sp1, and BK(Dunlop) contains two binding sites for transcription factor AP-1. The AP-1 sites in BK(Dunlop) span the junction of adjacent direct repeats, suggesting that repeat formation may be an important mechanism for de novo formation of binding sites not present in a parental strain.
Insulinlike growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers
Authors: Fu X X , Su C Y , Lee Y , Hintz R , Biempica L , Snyder R , Rogler C E
Published: January 2020

Insulinlike growth factor II (IGF-II) is a highly mitogenic fetal growth factor suspected of regulating the growth of a wide spectrum of tissues via an autocrine or paracrine mode of action or both. High steady-state levels of IGF-II RNA were detected in 45% of hepatocellular carcinomas (HCCs) arising from woodchuck livers with persistent woodchuck hepatitis virus (WHV) infection. Analysis of WHV RNA in the same HCCs revealed that HCCs with high levels of IGF-II RNA contained low or undetectable levels of WHV RNA and HCCs with low levels of IGF-II RNA contained high levels of WHV RNA. Integrated WHV DNA was present in HCCs from both groups, but viral DNA replicating forms were present, predominantly in HCCs with low levels of IGF-II. Several IGF-II RNAs, the most prominent of which were poly(A) species of approximately 3.75 and 1.1 to 1.3 kilobases, were detected only in precancerous nodules and HCCs. Levels of IGF-II were elevated two- to three-fold in the serum of woodchucks with chronic active hepatitis preceding the occurrence of HCC. Proliferation of a population of oval cells, which arise from portal tract regions in the liver, preceded the development of HCC and was a prominent feature of livers from which tumors with high levels of IGF-II occurred. The HCCs tended to have distinct histological features according to their growth factor status. Tumors with low levels of IGF-II were generally highly differentiated acinar-trabecular HCCs, whereas tumors with high levels of IGF-II were more anaplastic, with regions of fibrosis and fatty accumulation. A model to relate the pathology of WHV infection to oval cell proliferation and IGF-II expression in the development of these heterogeneous HCCs is presented.
Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus
Authors: Strynadka N C , Redmond M J , Parker J M , Scraba D G , Hodges R S
Published: January 2020

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.
Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis
Authors: McGuire T C , Norton L K , O'Rourke K I , Cheevers W P
Published: January 2020

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.
Latent herpes simplex virus type 1 transcription in human trigeminal ganglia
Authors: Steiner I , Spivack J G , O'Boyle D R , Lavi E , Fraser N W
Published: January 2020

We studied latent herpes simplex virus type 1 gene expression in human trigeminal ganglia. Two transcripts were mapped to a 3.0-kilobase region within the long repeat region and appeared to be located in neuronal nuclei. These viral RNAs were not abundant during lytic replication and may represent an alternative pattern of herpes simplex virus type 1 gene expression involved in the pathogenesis of latent infection.
Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells
Authors: Caughey B , Race R E , Ernst D , Buchmeier M J , Chesebro B
Published: January 2020

Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
Capped poly(A) leaders of variable lengths at the 5' ends of vaccinia virus late mRNAs
Authors: Ahn B Y , Moss B
Published: January 2020

Evidence for capped poly(A) leaders of variable lengths located immediately upstream of the translation initiation codon was obtained by direct analyses of a major late mRNA species. A decapping-recapping method was used to specifically substitute a radioactively labeled phosphate for an unlabeled one within the cap structure. RNase H-susceptible sites were made by hybridizing synthetic oligodeoxyribonucleotides to the mRNA encoding a late major structural protein of 11 kilodaltons. Sequences of the type m7G(5')pppAmp (Ap)nUpG. . ., where n varies from a few to more than 40 nucleotides, were deduced by analysis of the length and sequence of RNase H, RNase T1, and RNase U2 digestion products.
Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping
Authors: Papsidero L D , Sheu M , Ruscetti F W
Published: January 2020

Monoclonal antibodies (MAbs) to human immunodeficiency virus type 1 were produced. Two antibodies reacted with the 17-kilodalton core protein (p17) of the virus and with its polyprotein precursor. To various degrees, each MAb neutralized infection by the cell-free virus. With a series of sequential overlapping hexapeptides which represent the p17 gene product, the epitopes identified by the MAbs were defined. The epitopes localize to overlapping regions near the amino terminus of the protein. Soluble synthetic peptides which span the antibody-binding sites of interest were demonstrated to competitively inhibit the reactivity of p17 MAbs, thus confirming the location of virus-neutralizing sites within the core protein.
Comeasurement of simian virus 40 early and late promoter activity in HeLa and 293 cells in the presence of T antigen
Authors: Kelly J J , Munholland J M , Wildeman A G
Published: January 2020

Transcription of the simian virus 40 (SV40) late promoter is strongly activated by SV40 T antigen. We were interested in examining this process in relation to other T-antigen functions such as replication and repression of early transcription. To quantitate the various T-antigen effects, we used a vector which has a promoterless beta-globin gene flanking the early and late sides of the SV40 promoter region. Following cotransfection with a plasmid encoding T antigen, transcription from the two promoters can be measured with a single S1 mapping probe and replication can be assayed by Southern blot analysis of DNA recovered in Hirt extracts. In this study, transactivation was examined in HeLa and 293 cells, since these cells differ in their ability to support SV40 replication. The strength of the late promoter relative to the early promoter was approximately three- to fourfold higher in 293 cells. Replication in 293 cells was also more efficient, by the same margin. In both cell lines, late promoter transactivation was barely detectable on replication-defective templates. Taken together, the results suggest that T-antigen activation of late transcription occurs only on replicated, or replicating, DNA. T antigen also activated the late-early start sites, and while in HeLa cells they were seen to be only 30% as strong as the late promoter, in 293 cells late and late-early activities were almost equal.
Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro
Authors: Sarnow P
Published: January 2020

It has been shown by van der Werf et al. (S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier, and J. Dunn, Proc. Natl. Acad. Sci. USA 83:2330-2334, 1986) that in vitro synthesis of poliovirus RNA by T7 RNA polymerase gives rise to infectious RNA molecules; however, these molecules are only 5% as infectious as RNA isolated from virions. A plasmid, T7D-polio, was constructed that allows the in vitro synthesis of full-length RNA molecules with two additional guanine residues at the 5' end. However, T7D-polio differed from the construct of van der Werf et al. in that RNA transcribed from T7D-polio has an authentic 3' end, ending with only a polyadenine nucleotide sequence. Transfection of these RNA molecules into mammalian cells produced wild-type poliovirus with an efficiency similar to that of virion RNA. The use of this vector in the characterization of viral mutants in vivo and in vitro is discussed.
Epstein-Barr virus (EBV)-containing nasopharyngeal carcinoma cells express the B-cell activation antigen blast2/CD23 and low levels of the EBV receptor CR2
Authors: Billaud M , Busson P , Huang D , Mueller-Lantzch N , Rousselet G , Pavlish O , Wakasugi H , Seigneurin J M , Tursz T , Lenoir G M
Published: January 2020

Anaplastic nasopharyngeal carcinoma (NPC) cells invariably harbor the Epstein-Barr virus (EBV) genome, an association that is unique among human virus-associated cancers. Although EBV is able to replicate in epithelial cells, results with expression of the EBV receptor (complement receptor type 2 [CR2]; also called CD21) in normal and malignant epithelial cells are conflicting. We grew five different EBV-associated NPC tumors in nude mice, and by using a sensitive transcriptional assay, we detected a very weak transcription signal of the EBV receptor CR2 gene in these cells. This suggests that low levels of EBV receptor may be expressed by malignant epithelial nasopharyngeal cells. The gene coding for Blast2/CD23, a B-cell activation molecule induced by EBV, was transcribed in three of the transplanted NPC tumors. The soluble form of the Blast2/CD23 protein was also detected in medium taken from short-term cultures of the same NPC cell lines. In contrast to the lymphoid system, in which Blast2/CD23 expression is associated with EBV nuclear antigen (EBNA2) expression, no EBNA2 protein could be detected in these NPC epithelial cells. Our study represents the first demonstration of Blast2/CD23 expression in epithelial cells. As the soluble form of the Blast2/CD23 protein possesses growth factor activity associated with EBV-induced B-cell immortalization, these results suggest a possible role for this molecule in the pathogenesis of NPC.
Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication
Authors: Mohr I J , Fairman M P , Stillman B , Gluzman Y
Published: January 2020

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.
Theiler's murine encephalomyelitis virus neutralization escape mutants have a change in disease phenotype
Authors: Roos R P , Stein S , Routbort M , Senkowski A , Bodwell T , Wollmann R
Published: January 2020

DA strain of Theiler's murine encephalomyelitis virus produces a persistent demyelinating infection. We previously produced escape mutant viruses that are resistant to a neutralizing monoclonal antibody and have a mutation in VP1 amino acid residue 268 in a neutralization site (Y. Ohara, A. Senkowski, J. Fu, L. Klaman, J. Goodall, M. Toth, and R.P. Roos, J. Virol. 62:3527-3529, 1988). In contrast to wild-type DA strain, these escape mutants produce little if any demyelinating disease after inoculation into weanling mice.
Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene
Authors: Zhu J Y , Cole C N
Published: January 2020

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.
The herpes simplex virus virion host shutoff function
Authors: Kwong A D , Frenkel N
Published: January 2020

The virion host shutoff (vhs) function of herpes simplex virus (HSV) limits the expression of genes in the infected cells by destabilizing both host and viral mRNAs. vhs function mutants have been isolated which are defective in their ability to degrade host mRNA. Furthermore, the half-life of viral mRNAs is significantly longer in cells infected with the vhs-1 mutant virus than in cells infected with the wild-type (wt) virus. Recent data have shown that the vhs-1 mutation resides within the open reading frame UL41. We have analyzed the shutoff of host protein synthesis in cells infected with a mixture of the wt HSV-1 (KOS) and the vhs-1 mutant virus. The results of these experiments revealed that (i) the wt virus shutoff activity requires a threshold level of input virions per cell and (ii) the mutant vhs-1 virus protein can irreversibly block the wt virus shutoff activity. These results are consistent with a stoichiometric model in which the wt vhs protein interacts with a cellular factor which controls the half-life of cell mRNA. This wt virus interaction results in the destabilization of both host and viral mRNAs. In contrast, the mutant vhs function interacts with the cellular factor irreversibly, resulting in the increased half-life of both host and viral mRNAs.
Derivation and characterization of an efficiently myocarditic reovirus variant
Authors: Sherry B , Schoen F J , Wenske E , Fields B N
Published: January 2020

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.
The reovirus M1 gene, encoding a viral core protein, is associated with the myocarditic phenotype of a reovirus variant
Authors: Sherry B , Fields B N
Published: January 2020

Reoviruses contain a genome composed of 10 double-stranded RNA gene segments. A reovirus reassortant, 8B, derived from type 1 Lang (T1L) and type 3 Dearing (T3D), displayed a phenotype unlike that of either of its parents in that it efficiently induced numerous macroscopic external cardiac lesions in neonatal mice (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989). A panel of T1L/T3D reassortants and a panel of reassortants derived from 8B were used to determine whether novel T1L/T3D gene associations in 8B were responsible for its myocarditic phenotype. The results eliminated the possibility that any T1L/T3D gene combination found in 8B, from 2 genes to all 10 genes, was the explanation for its induction of cardiac lesions. This suggested that a mutation(s) in an 8B gene(s) might be responsible for induction of the myocarditis. Statistical analysis of experiments with 31 reassortants derived from 8B revealed a highly significant association (P = 0.002) of the 8B M1 gene with induction of cardiac lesions. The reovirus M1 gene encodes a viral core protein of unknown function, although evidence suggests a potential role in core structure and/or viral RNA synthesis. This represents the first report of the association of a viral gene with induction of myocarditis.
Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage
Authors: Burns C C , Lawson M A , Semler B L , Ehrenfeld E
Published: January 2020

A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.
Inducible expression of encephalomyocarditis virus 3C protease activity in stably transformed mouse cell lines
Authors: Lawson T G , Smith L L , Palmenberg A C , Thach R E
Published: January 2020

An inducible expression vector system has been developed to facilitate the study of the effects of individual virus gene products on cell function. The vector utilizes the mouse metallothionein promoter carried on the bovine papillomavirus genome. Conditions which optimize the induced expression of open reading frames inserted downstream from the mouse metallothionein promoter have recently been described. In this communication we describe the use of this system to clone and express the encephalomyocarditis virus 3C protease in cultured mouse cells. Stably transformed cell lines could be induced to produce levels of 3C protease activity comparable to those observed during normal virus infection. In spite of this, no effects on cellular protein synthesis rate or membrane permeability were observed. It was also discovered that 3C protease as well as 3C protease-containing polyproteins are turned over. This was true not only in the induced cell clones, but also during the normal course of encephalomyocarditis virus infection, as well as in translation systems in vitro. This phenomenon was highly specific for this family of polypeptides, perhaps explaining their apparent lack of cytotoxic effects.
The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase
Authors: Gallo M L , Dorsky D I , Crumpacker C S , Parris D S
Published: January 2020

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.
Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120
Authors: Arthur L O , Bess J W , Waters D J , Pyle S W , Kelliher J C , Nara P L , Krohn K , Robey W G , Langlois A J , Gallo R C
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.
Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2
Authors: O'Neill R E , Racaniello V R
Published: January 2020

A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.
Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1
Authors: McBride A A , Bolen J B , Howley P M
Published: January 2020

The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes three transcriptional regulatory proteins. The full-length open reading frame encodes a protein of 410 amino acids which functions as a transcriptional transactivator. Two transcriptional repressor proteins, E2-TR and E8/E2, contain the C-terminal 249 and 204 amino acids, respectively. We have expressed both the full-length E2 protein and the E2-TR repressor protein in insect cells, by using recombinant baculoviruses, and in mammalian COS-1 cells, by using a chimeric simian virus 40/BPV-1 virus. Analysis of the E2 proteins revealed that both the transactivator and repressor forms are phosphorylated predominately on serine residues at similar sites in both expression systems. By a combination of peptide mapping and site-directed mutagenesis techniques, the serine residues at positions 298 and 301 were determined to be the major phosphorylation sites of the BPV-1 E2 proteins.
The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration
Authors: Katzman M , Katz R A , Skalka A M , Leis J
Published: January 2020

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.
Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene
Authors: Klucher K M , Rabert D K , Spector D H
Published: January 2020

We have studied the regulation of expression of a major human cytomegalovirus (HCMV) early transcription unit located within the long repeat of the strain AD169 genome. This region specified a 2.7-kilobase RNA which underwent its largest increase in abundance between 8 and 14 h postinfection. To study the regulation of this gene, its promoter was cloned 5' of the gene for chloramphenicol acetyltransferase (CAT) for use in transient expression assays. A construct containing 651 base pairs of upstream sequence and 54 base pairs of leader sequence was transfected into human fibroblast cells, followed by HCMV infection. Analysis of the steady-state levels of RNA expressed from this hybrid gene indicated that it accumulated with the same kinetics as the authentic viral transcript early in the infection. Cotransfection of human fibroblasts with the 2.7-kilobase RNA promoter-CAT construct and plasmids containing different HCMV immediate-early (IE) genes showed that the region of the HCMV genome encoding the transcription units corresponding to IE1 and 2 and the 5' end of IE3 is capable of stimulating promoter activity but not to the same extent as HCMV infection. To define important cis-acting regulatory elements in the promoter, a series of 5' deletion mutants was constructed. Transient expression analysis showed a stepwise reduction in inducible CAT activity, suggesting the presence of multiple regulatory sites. To further characterize the nature of these sites, we used gel mobility shift assays to study DNA-protein interactions occurring within this promoter sequence. With nuclear extracts prepared from HeLa cells as well as from infected and uninfected human foreskin fibroblasts, we found specific binding of a cellular factor to a region of the promoter important in HCMV inducible activity. This region contains a palindromic octamer with homology to the binding site of the cellular factor USF/MLTF.
Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome
Authors: Iizuka N , Kohara M , Hagino-Yamagishi K , Abe S , Komatsu T , Tago K , Arita M , Nomoto A
Published: January 2020

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.
Peripheral T-lymphocyte activation by human T-cell leukemia virus type I interferes with the CD2 but not with the CD3/TCR pathway
Authors: Dodon M D , Bernard A , Gazzolo L
Published: January 2020

Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukemia, an aggressive lymphoproliferative disorder, and with chronic neurological diseases. In vitro it can infect several types of cells but transforms only human T lymphocytes. We have previously shown that HTLV-I viral particles, even when noninfectious, were able to activate human resting T lymphocytes, suggesting that this activation step may be important in the initiation of the lymphoproliferative process. In the present study, we first demonstrate that in contrast to other mitogenic stimuli, HTLV-I has the unique property to activate human resting T cells in the absence of accessory cells. We then investigate the relationship between HTLV-I-induced T-cell activation and the classical well-known pathways of activation, namely, the CD3/TCR and CD2 pathways. Competitive blocking experiments were performed in which the effects of monoclonal antibodies (MAb) to the CD3/TCR complex or to the CD2 molecule were evaluated on the HTLV-I activation of T cells and compared with that obtained on phytohemagglutinin (PHA)-stimulated cells. It was found that anti-CD3 or -TCR MAb strongly suppress the proliferative response of T cells to PHA, but are significantly less efficient in inhibiting the activation initiated by HTLV-I. By contrast, MAb recognizing specific epitopes of the CD2 molecule inhibit the proliferative response of T cells to PHA or to HTLV-I to the same extent. The results provide evidence that HTLV-I virions interfere mainly with activation via CD2 but not via the CD3/TCR complex. Considering the earlier expression of the CD2 molecule on human T-cell precursors, these observations might be relevant to the characterization of the differentiation stage at which viral infection could interfere with the development and the maturation of T lymphocytes.
Permanent cell lines that show temperature-dependent expression of adenovirus virus-associated RNA
Authors: Martínez C , de la Luna S , Peláez F , Lopez-Turiso A , Valcárcel J , de Haro C , Ortín J
Published: January 2020

Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection.