bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous rearrangement of integrated simian virus 40 DNA in nine transformed rodent cell lines
Authors: Gurney T , Gurney E G
Published: January 2020

Frequencies of spontaneous DNA rearrangement within or near integrated simian virus 40 (SV40) DNA were measured in four transformed mouse and rat cell lines of independent origin and in five clones of the SV40-transformed mouse line SVT2. Rearrangements were detected as polymorphisms of restriction enzyme fragment length in subclones of the lines. At least 17% of the subclones of each line had detectable rearrangements. The rate of rearrangement was calculated to be at least 5 x 10(-3) events per cell per division. No rearrangements were detected in sequences of an immunoglobulin gene, part of the coding region of the mouse protein p53, and five proto-oncogenes. The possible role of recombination between duplicated segments of integrated SV40 DNA in generating rearrangements was studied in the five SVT2 clones, which differed in the number of duplications within a single SV40 DNA segment. The SVT2 clone that had no duplications, M3, became rearranged further at least as frequently as did closely related lines with one, two, or three duplications. Another line in this group that had one small duplication, X1, had a much higher frequency of rearrangement than did the others; integrated SV40 DNA of X1 became mostly rearranged within 100 cell divisions. The examples of M3 and X1 suggested that the high rate of rearrangement characteristic of integrated SV40 DNA was influenced more by the presence of particular sequences within or near integrated SV40 DNA than by the number or extent of duplicated sequences.
Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors
Authors: Hara T , Hattori S , Kawakita M
Published: January 2020

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.
Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression
Authors: Olivo P D , Nelson N J , Challberg M D
Published: January 2020

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.
Effect of mutations and deletions in a bicistronic mRNA on the synthesis of influenza B virus NB and NA glycoproteins
Authors: Williams M A , Lamb R A
Published: January 2020

The mRNA derived from influenza B virus RNA segment 6 is functionally bicistronic and encodes the NB and NA glycoproteins in different, overlapping reading frames. NB protein synthesis is initiated at the 5'-proximal AUG codon, and 4 nucleotides downstream there is a second AUG codon which is used to initiate NA protein synthesis. The nucleotide sequence context of the first AUG codon conforms closely with the established 5'-CC(A/G)CCAUGG-3' consensus sequence (M. Kozak, Nucleic Acids Res. 15:8125-8148, 1987), which should favor initiation of NB protein synthesis at this site, yet NB and NA are found to accumulate in approximately equal amounts in infected cells. To determine the features important for allowing initiation at the second 5'-proximal AUG codon, we made changes in the 5'-terminal region of the mRNA, including deletions, insertions, and site-specific mutations. The recombinant DNA molecules were expressed in eucaryotic cells, and the accumulation of NB and NA was quantitated. The data indicate that changes in the immediate sequence around the first AUG codon do not make a large difference in the amounts of NB and NA that accumulate, but that when the first AUG codon is displaced from its normal position it is now quite efficient at preventing downstream initiation events. In addition, the data indicate that an element of the B/NB/NA mRNA 5' untranslated leader region acts in cis to enhance the expression of NB and NA.
Distinct segments within the enhancer region collaborate to specify the type of leukemia induced by nondefective Friend and Moloney viruses
Authors: Golemis E , Li Y , Fredrickson T N , Hartley J W , Hopkins N
Published: January 2020

The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)
A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner
Authors: Verderame M F , Kaplan J M , Varmus H E
Published: January 2020

The v-src oncogene of Rous sarcoma virus (RSV) is able to transform both avian and mammalian cells, but the mutant allele v-src-L displays a host range dependence for transformation, transforming chicken but not rat cells with wild-type efficiency. This host range restriction can be detected by measuring growth in low serum, saturation density, and anchorage independent growth. In addition, rat cells expressing v-src-L do not form tumors in syngeneic rats or nude mice, but RSV carrying the mutant allele causes tumors in chicks, although at a reduced efficiency and with increased latency. To determine the lesion responsible for this phenotype, we sequenced the entire v-src gene from the parental B77 strain of RSV, as well as the mutant allele. v-src-L is missing 3 nucleotides present in the wild-type parent, RSV B31, eliminating Phe-172, an invariant residue in a conserved region of src-related proteins known as SH-2. The kinase activity of pp60v-src-L was indistinguishable from that of the wild type in chicken cells but was significantly reduced in rat cells as assayed by an in vitro immune complex assay; in vivo phosphorylation of one specific substrate, p36 (calpactin I heavy chain); and total phosphotyrosine-containing proteins. In addition, the pattern of phosphotyrosine-containing proteins in rat cells was qualitatively different when cells containing pp60v-src-L were compared with cells with wild-type pp60v-src, even though both pp60v-src proteins were membrane associated. The data are consistent with a role for the SH-2 region in substrate specificity.
Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus
Authors: Olmsted R A , Murphy B R , Lawrence L A , Elango N , Moss B , Collins P L
Published: January 2020

Posttranslational processing and cell surface expression were examined for three C-terminally truncated mutants of the G protein of respiratory syncytial virus expressed from engineered cDNAs. The truncated mutants, encoded by cDNAs designated G71, G180, and G230, contained the N-terminal 71, 180, and 230 amino acids, respectively, of the 298-amino-acid G protein. To facilitate detection of G71, which reacted inefficiently with G-specific antisera, we constructed a parallel set of cDNAs, designated G71/13, G180/13, and G230/13, to encode the same truncated species with the addition of a C-terminal 13-amino-acid reporter peptide which could be detected efficiently with an antipeptide serum. G71, G180, and G230 were expressed as species of Mr 7,500, 48,000, and 51,000, respectively, compared with 84,000 for parental G protein. The proteins encoded by G180 and G230, like parental G protein, contained both N-linked and O-linked carbohydrate. Also, the protein encoded by G71/13 appeared to be O glycosylated, showing that even this highly truncated form contained the structural information required to target the protein for O glycosylation. As for parental G protein, the estimated Mrs of the proteins encoded by G180 and G230 were approximately twice the calculated molecular weight of the polypeptide chain. Experiments with monensin showed that most of this difference between the calculated and observed Mr was due to posttranslational processing in or beyond the trans-Golgi compartment, presumably owing to the addition of carbohydrate or aggregation into dimers or both. Like parental G protein, all three truncated forms accumulated abundantly at the cell surface, and in each case the C terminus was extracellular. Thus, the N-terminal 71 amino acids of the G protein contained all the structural information required for efficient membrane insertion and cell surface expression, whereas the extracellular domain was dispensable for these activities. Cotton rats were immunized with recombinant vaccinia viruses expressing the G71, G180, G230, or parental G protein to compare their abilities to induce serum antibodies and resistance to challenge virus replication. The G71 and G180 recombinants failed to induce significant levels of G-specific antibodies or resistance to challenge, whereas the immunogenicity of G230 equaled or exceeded that of parental G protein. This suggested that the C-terminal 68 amino acids of the 236-amino-acid ectodomain do not contribute to the major epitope(s) of the G protein that is involved in inducing protective immunity.
Amplification of mouse mammary tumor virus genomes in non-mammary tumor cells
Authors: Racevskis J , Beyer H
Published: January 2020

Extra proviral copies of mouse mammary tumor virus (MMTV) are known to be present in the genomes of certain T-cell lymphomas of mice. Analysis of additional non-mammary tumor cell types known to express MMTV transcripts and antigens revealed the presence of extra acquired MMTV proviruses in a pituitary tumor cell line, a macrophage line, and Leydig testicular tumor cells. The nature of the amplified MMTV proviruses in these various tumor cell types differed with regard to copy number and presence of alterations in the long terminal repeat region.
Initial interaction of herpes simplex virus with cells is binding to heparan sulfate
Authors: WuDunn D , Spear P G
Published: January 2020

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.
Identification of factors specific for transcription of the late class of vaccinia virus genes
Authors: Wright C F , Moss B
Published: January 2020

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
Treatment of encephalomyocarditis virus-induced central nervous system demyelination with monoclonal anti-T-cell antibodies
Authors: Sriram S , Topham D J , Huang S K , Rodriguez M
Published: January 2020

Infection of BALB/c mice with the M variant of encephalomyocarditis virus resulted in the development of a paralytic syndrome in 7 to 10 days. The paralysis was maximal during the period of viral clearance; most of the animals recovered from the initial deficit and showed no delayed recurrences. Pathologically, the white matter of brain and spinal cord showed well-demarcated areas of perivascular cuffing, demyelination, and, during recovery, remyelination by oligodendrocytes--all suggestive of postinfectious encephalomyelitis. Depletion of either the CD4 or CD8 subset of T cells in vivo with the appropriate monoclonal antibody, GK1.5 or 2.43, respectively, administered one day (24 h) prior to infection was sufficient to limit the development of the paralytic syndrome by 79% (GK1.5) and 82% (2.43).
Cloned hepatitis delta virus cDNA is infectious in the chimpanzee
Authors: Sureau C , Taylor J , Chao M , Eichberg J W , Lanford R E
Published: January 2020

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.
Molecularly engineered vaccine which expresses an immunodominant T-cell epitope induces cytotoxic T lymphocytes that confer protection from lethal virus infection
Authors: Klavinskis L S , Whitton J L , Oldstone M B
Published: January 2020

Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.
The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes
Authors: Bernard B A , Bailly C , Lenoir M C , Darmon M , Thierry F , Yaniv M
Published: January 2020

The human papillomavirus type 18 (HPV18) long control region (LCR) harbors transcriptional promoter and enhancer elements. Recombinant plasmids bearing all or part of the HPV18 LCR cloned in enhancer or promoter configuration upstream of the chloramphenicol acetyltransferase (CAT) gene were transfected into human fibroblasts and keratinocytes. Although the HPV18 enhancer can function in the absence of E2 gene products in both fibroblasts and keratinocytes, the promoter activity of the HPV18 LCR is detectable in keratinocytes but not in fibroblasts, suggesting that it is tissue specific. This promoter activity was repressed in human keratinocytes not only by the bovine papillomavirus type 1 E2 gene product but also by the homologous HPV18 E2 gene product. The promoter involved in the HPV18 E2 repression is located within a 230-base-pair domain directly upstream of the E6 open reading frame of the HPV18 LCR and is probably the previously identified E6 promoter. Although one cannot rule out the possibility that this repressing effect is mediated by a truncated form of HPV18 E2 protein, as was previously demonstrated for bovine papillomavirus type 1, a more likely explanation would be that the full-length HPV18 E2 protein behaves as a repressor. Indeed, at the same doses at which it inhibits transcription from the homologous HPV18 LCR, the HPV18 E2 gene product activates transcription from constructs bearing E2-binding palindromes cloned in enhancer configuration upstream of a heterologous promoter. The fact that the homologous HPV18 E2 gene product acts as a transcriptional repressor of the HPV18 LCR suggests a possible explanation for the overexpression of E6 and E7 open reading frames in cervical carcinoma cells and in cell lines derived from them.
Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells
Authors: Wills J W , Craven R C , Achacoso J A
Published: January 2020

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.
The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells
Authors: Bodnar J W , Hanson P I , Polvino-Bodnar M , Zempsky W , Ward D C
Published: January 2020

The interaction of viral genomes with the cellular nuclear matrix was studied by using adenovirus-infected HeLa cells and minute virus of mice (MVM)-infected A-9 cells. Adenovirus DNA was associated with the nuclear matrix both early and late in infection, the tightest interaction being with DNA fragments that contain the covalently bound 5'-terminal protein. Replicative forms of MVM DNA were also found to be exclusively matrix associated during the first 16 to 20 h of infection; at later times viral DNA species accumulated in the soluble nuclear fraction at different rates, suggesting a saturation of nuclear matrix-binding sites. MVM DNA fragments enriched in the matrix fraction were also derived from the terminal regions of the viral genome. However, only the subset of fragments which possess a covalently bound 5'-terminal protein (i.e., DNA fragments in which the 5' palindromic DNA sequences are in the extended duplex rather than the hairpin conformation) were matrix associated. These observations suggest that the DNA-matrix interactions are, at least in part, mediated by the viral terminal proteins. Since these proteins have previously been shown to be intimately involved in viral DNA replication, our results further indicate that an association with the nuclear matrix may be important for viral genome replication and possibly also for efficient gene transcription.
Characterization of in vitro inhibition of human immunodeficiency virus by purified recombinant CD4
Authors: Byrn R A , Sekigawa I , Chamow S M , Johnson J S , Gregory T J , Capon D J , Groopman J E
Published: January 2020

The first step in infection of human T cells with human immunodeficiency virus (HIV) is binding of viral envelope glycoprotein gp120 to its cellular receptor, CD4. The specificity of this interaction has led to the development of soluble recombinant CD4 (rCD4) as a potential antiviral and therapeutic agent. We have previously shown that crude preparations of rCD4 can indeed block infection of T cells by HIV type 1 (HIV-1). Here we present a more detailed analysis of this antiviral activity, using HIV-1 infection of the T lymphoblastoid cell line H9 as a model. Purified preparations of rCD4 blocked infection in this system at nanomolar concentrations; combined with the known affinity of the CD4-gp120 interaction, this finding suggests that the inhibition is simply due to competition for gp120 binding. As predicted, rCD4 had comparable activity against all strains of HIV-1 tested and significant activity against HIV-2. Higher concentrations of rCD4 blocked infection even after the virus had been adsorbed to the cells. These findings imply that the processes of viral adsorption and penetration require different numbers of gp120-CD4 interactions. Recombinant CD4 was able to prevent the spread of HIV infection in mixtures of uninfected and previously infected cells. Our studies support the notion that rCD4 is a potent antiviral agent, effective against a broad range of HIV-1 isolates, and demonstrate the value of purified rCD4 as an experimental tool for studying the mechanism of virus entry into cells.
Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro
Authors: Carrington J C , Freed D D , Sanders T C
Published: January 2020

The virus-encoded proteins of tobacco etch virus (TEV), a plant potyvirus, arise by proteolytic processing of a large polyprotein precursor. The TEV genome codes for two proteinases, a 49-kilodalton proteinase and helper component proteinase (HC-Pro), which cleave the polyprotein at specific sites. The only known cleavage event catalyzed by HC-Pro occurs at the HC-Pro carboxyl terminus. The proteolytic activity of HC-Pro was analyzed by expression of the enzyme in bacterial and cell-free systems. The carboxyl-terminal domain of HC-Pro exhibited proteolytic activity in Escherichia coli with a processing half-time of approximately 100 s. The processing kinetics of HC-Pro expressed in vitro by cell-free transcription and translation was variable, depending on the presence or absence of TEV polypeptide sequences at the amino terminus of the proteolytic domain. Cleavage of the HC-Pro carboxyl terminus appeared to proceed exclusively by an autocatalytic mechanism; the proteinase synthesized in vitro exhibited little or no proteolytic activity when reacted with the HC-Pro cleavage site in trans or biomolecular reactions.
The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins
Authors: Pallas D C , Morgan W , Roberts T M
Published: January 2020

We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.
The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule
Authors: Hardwicke M A , Vaughan P J , Sekulovich R E , O'Conner R , Sandri-Goldin R M
Published: January 2020

The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.
Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics
Authors: Kawaoka Y , Krauss S , Webster R G
Published: January 2020

We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.
Use of Ar+ plasma etching to localize structural proteins in the capsid of herpes simplex virus type 1
Authors: Newcomb W W , Brown J C
Published: January 2020

Partially cored herpes simplex virus type 1 (HSV-1) capsids (B capsids) were eroded in a low-energy (0.5-keV) Ar+ ion plasma under conditions in which the outermost structural proteins were expected to be degraded before more internal ones. After various periods of etching, the proteins remaining intact were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined quantitatively by densitometric scanning of the stained gels. The results showed that the major capsid polypeptide (VP5) and two other capsid proteins, VP19 and VP23, were degraded rapidly beginning as soon as capsids were exposed to the ion plasma. In contrast, significant lags were observed for erosion of VP21, VP22a, and VP24, suggesting that these proteins were available to accelerated ions only after other, more external structures had been damaged or eroded away. The results suggest that VP5, VP19, and VP23 are exposed on the surface of the capsid, while VP21, VP22a, and VP24 are found inside the capsid cavity. The experiments are consistent with the view that VP5 constitutes the major structural component of the hexavalent capsomers. It is proposed that VP19 and VP23 may form other surface structures such as the pentavalent capsomers, the capsid floor, or the intercapsomeric fibers.
Significance of premature stop codons in env of simian immunodeficiency virus
Authors: Kodama T , Wooley D P , Naidu Y M , Kestler H W , Daniel M D , Li Y , Desrosiers R C
Published: January 2020

The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIVmac). The SIVmac251 and SIVmac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIVmac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). At 20 to 30 days posttransfection, SIVmac239 virus with a 41-kDa TMP gradually disappeared coincident with the emergence of a virus with a 28-kDa TMP. Virus production dramatically increased in parallel with the emergence of a virus with a 28-kDa TMP. Sequence analysis of viral DNAs from these cultures showed that premature stop codons arising by point mutation were responsible for the change in size of the TMP with time. A similar selective pressure for truncated forms of TMP was observed when the SIVmac239 clone was transfected into human peripheral blood lymphocytes (PBL). In contrast, no such selective pressure was observed in macaque PBL. When the SIVmac239 clone was transfected into macaque PBL and the resultant virus was serially passaged in macaque PBL, the virus replicated very well and maintained a 41-kDa TMP for 80 days in culture. Macaque monkeys were infected with SIVmac239 having a 28-kDa TMP; virus subsequently recovered from T4-enriched lymphocytes of peripheral blood showed only the 41-kDa form of TMP. These results indicate that the natural form of TMP in SIVmac is the full-length 41-kDa TMP, just as in human immunodeficiency virus type 1. Viruses with truncated forms of TMP appear to result from mutation and selection during propagation in unnatural human cells.
Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis
Authors: Maione R , Felsani A , Pozzi L , Caruso M , Amati P
Published: January 2020

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.
Human papillomavirus type 49, a type isolated from flat warts of renal transplant patients
Authors: Favre M , Obalek S , Jablonska S , Orth G
Published: January 2020

The cloning and characterization of the genome of human papillomavirus type 49 (HPV-49) is described. The viral DNA, which is most closely related to the DNAs of HPVs seen in patients with epidermodysplasia verruciformis, was aligned to the HPV-5 genome by electron microscopic analysis of heteroduplexes between the cloned viral DNAs.
Analysis of regulatory elements of the equine infectious anemia virus and caprine arthritis-encephalitis virus long terminal repeats
Authors: Sherman L , Yaniv A , Lichtman-Pleban H , Tronick S R , Gazit A
Published: January 2020

We analyzed the equine infectious anemia virus (EIAV) long terminal repeat (LTR) for sequences that influence its promoter activity and ability to be trans-activated by the EIAV tat gene product. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 (SV40) regulatory sequences were used for these studies. We were able to identify the EIAV promoter region and showed that sequences within the U3 region significantly inhibited LTR-directed transcription. However, when placed in a heterologous context (SV40 promoter) these U3 sequences functioned as an enhancer. trans-activation of the EIAV LTR was found to depend upon sequences downstream of the transcription initiation site and also within U3. Deletion mutagenesis experiments showed that the major downstream element was present in a 46-nucleotide stretch (+4 to +50). An SV40 promoter construct containing these sequences could be trans-activated in cells expressing the EIAV tat gene product. For comparative purposes we also examined the LTR of another animal lentivirus, caprine arthritis-encephalitis virus (CAEV), for positive and negative transcriptional regulatory elements and demonstrated the presence of an enhancer within its U3 sequence. There is evidence that trans-activation of the CAEV LTR requires U3 sequences. When the EIAV U3 region was replaced by the CAEV U3 sequence, the promoter activity of the EIAV LTR was markedly elevated, but the responsiveness to the EIAV trans-activator could not be restored.
Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV
Authors: Brown E A , Jansen R W , Lemon S M
Published: January 2020

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.
Mutational analysis of bovine papillomavirus type 1 E5 peptide domains involved in induction of cellular DNA synthesis
Authors: Rawls J A , Loewenstein P M , Green M
Published: January 2020

Early gene E5 of bovine papillomavirus type 1 encodes a 44-amino-acid protein whose expression can transform immortalized mouse cell lines. We have previously reported that a chemically synthesized E5 peptide functions to induce cellular DNA synthesis upon microinjection into growth-arrested mouse cells. We further defined the two E5 domains essential for the full DNA synthesis induction activity by the analysis of E5 deletion and amino acid substitution mutant peptides. The first domain is the C-terminal 13-amino-acid core which is sufficient to activate DNA synthesis at high peptide concentration and contains two essential, highly conserved cysteine residues. The second domain is the 7-amino-acid hydrophobic sequence contiguous to the core domain which is sufficient to confer a 1,000-fold higher molar specific activity to the E5 peptide. A random hydrophobic sequence, but not charged amino acids, fulfills the function of the second domain.
Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells
Authors: Haddad R S , Hutt-Fletcher L M
Published: January 2020

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.