bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of envelope glycoprotein processing in murine leukemia virus infection
Authors: Freed E O , Risser R
Published: January 2020

The murine leukemia virus envelope protein is synthesized as a precursor molecule, Pr85env, which is proteolytically cleaved at an arginine residue to produce two mature envelope proteins, gp70 and p15(E). The results presented here indicate that mutation to lysine of the arginine found at the envelope precursor cleavage site results in a precursor which is cleaved with an efficiency at least 10-fold lower than the efficiency with which the wild-type protein is cleaved. This mutation has been used to investigate the requirement for envelope protein processing in various aspects of retroviral infection. Viruses produced by cells transfected with mutant proviral clones are approximately 10-fold less infectious than wild-type viruses. Mutant viruses are incapable of inducing XC cell syncytium formation and are 100-fold less efficient than wild-type viruses at rendering cells resistant to superinfection. Envelope glycoproteins bearing the lysine mutation are found in reduced amounts on the surface of infected cells, and as a result mutant virions contain significantly less envelope protein than do wild-type virions. The phenotypic effects of the processing mutation described here are most likely the result of this paucity of envelope glycoproteins in virions carrying the mutation.
Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes
Authors: Stoye J P , Coffin J M
Published: January 2020

Inbred mice contain three classes of endogenous nonecotropic murine leukemia virus-related sequences, namely xenotropic, polytropic, and modified polytropic proviruses. Oligonucleotide probes specific for the three different classes were prepared and used to examine the diversity of endogenous sequences present in eight different strains of mice: HRS/J, BALB/cJ, A/J, AKR/J, C57BL/6J, DBA/2J, C57L/J, and C3H/HeJ. A high degree of polymorphism was observed. Overall, the strains showed between 17% (A/J and HRS/J) and 65% (C57BL/6J and C57L/J) shared proviruses, and only four proviruses were present in all eight strains. The similarity among the strains is due in part to the few proviruses present in all of the strains but also represents the independent assortment of a limited set of proviruses. These oligonucleotides provide a basis for determining the stability, distribution, and mutagenic potential of nonecotropic proviruses within the mouse genome.
Production of a polyhedral particle in Escherichia coli from a cDNA copy of the large genomic segment of bacteriophage phi 6
Authors: Gottlieb P , Strassman J , Bamford D H , Mindich L
Published: January 2020

A polyhedral particle that resembles in composition and structure the procapsid of bacteriophage phi 6 was produced in Escherichia coli containing cDNA copies of the entire large genomic segment inserted into expression vector plasmids under the control of lac or tac promoters. The particles were composed of proteins P1, P2, P4, and P7 in the same stoichiometry as in the intact virion. In electron micrographs of negatively stained samples, the particles appeared as hexagons, stars, or rings of 10 knobs, which are characteristic of the five-, three-, and twofold axes of symmetry characteristic of phi 6 procapsids. Stable particles were also produced from cDNA deletions that produce only P1 and P4. Other cDNA deletions producing P1 and P7 and P1 alone resulted in unstable particles which could only be visualized in electron micrographs of thin sections of E. coli transformed by the recombinant plasmids. Our results indicate that the assembly of the phi procapsid is independent of other phage proteins and of normal phage RNA.
Avian sarcoma-leukosis virus pol-endo proteins expressed independently in mammalian cells accumulate in the nucleus but can be directed to other cellular compartments
Authors: Morris-Vasios C , Kochan J P , Skalka A M
Published: January 2020

Eucaryotic expression vectors have been used to study transient expression of the avian sarcoma-leukosis retrovirus pol-endo protein in COS cells. The constructs encode proteins with N termini identical to that of authentic viral pp32 endonuclease with the exception of a single met residue encoded by the initiator AUG. The C termini correspond to unprocessed viral pol protein, authentic processed pp32, or a derivative which includes eight amino acids from the unprocessed portion. All three proteins localize to the nucleus. However, when the pol-endo domain is fused to a secretory signal peptide, the protein is found in medium and appears also to localize in the Golgi bodies and the cell membrane. These and derivative vectors will make it possible to assess the consequence of retroviral pol gene expression in eucaryotic cells.
Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus
Authors: Glickman R L , Syddall R J , Iorio R M , Sheehan J P , Bratt M A
Published: January 2020

Newcastle disease virus exhibits a wide range of pathogenicity and virulence which, as with all paramyxoviruses, is directly related to the cleavability of a precursor (F0) of the fusion glycoprotein by cellular proteases. Sequence analyses of the cleavage site of several virulent and avirulent isolates of the Newcastle disease virus serotype reveal a correlation between virulence or pathogenicity and a high content of basic amino acid residues at the cleavage site. A similar correlation has been seen for other paramyxoviruses.
An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation
Authors: Enquist L W , Keeler C L , Robbins A K , Ryan J P , Whealy M E
Published: January 2020

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.
Effects of anti-gp120 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1
Authors: Linsley P S , Ledbetter J A , Kinney-Thomas E , Hu S L
Published: January 2020

Monoclonal antibodies (MAbs) to defined peptide epitopes on gp120 from human immunodeficiency virus type 1 were used to investigate the involvement of their epitopes in gp120 binding to the CD4 receptor. Recombinant vaccinia viruses were constructed that expressed either full-length gp120 (v-ED6), or a truncated gp120 lacking 44 amino acids at the carboxyl terminus (v-ED4). Binding of these glycoproteins to the CD4 receptor was detected directly with metabolically labeled gp120 or indirectly with the gp120 MAbs. Truncated gp120 from v-ED4 bound to CD4-positive cells less than 1/12 as well as gp120 from v-ED6, indicating that the C-terminal region of gp120, which is conserved in numerous isolates of human immunodeficiency virus type 1, is critical for CD4 binding. However, MAb 110-1, which recognizes a peptide contained in the region deleted from v-ED4 (amino acids 489 through 511), did not inhibit binding of gp120 to CD4. MAb 110-1 also reacted with gp120 bound to the CD4 receptor, indicating that the epitope for this antibody does not directly interact with CD4. A second MAb, 110-4, which recognizes a peptide epitope located between amino acids 303 and 323 and has potent viral neutralizing activity, also bound to gp120 on the CD4 receptor. Furthermore, pretreatment of gp120 with MAb 110-4 at concentrations approximately 1,000-fold higher than those required for complete virus neutralization inhibited subsequent CD4 binding by only about 65%. Taken together, these data suggest that neutralization mediated by antibody 110-4 does not result from binding of this MAb to the CD4-binding site of gp120.
Duplication of noncoding sequences in polyomavirus specifically augments the development of thymic tumors in mice
Authors: Freund R , Dawe C J , Benjamin T L
Published: January 2020

A 40-base-pair duplication of noncoding sequences in polyomavirus specifically augmented the development of thymic epitheliomas following inoculation of virus into newborn mice. Virus strains carrying only one copy of this sequence induced a full spectrum of tumors except for overt thymic tumors. This 40-base-pair repeat, on the early side of the replication origin, constituted a tissue-specific regulatory determinant for tumor induction.
Fim-1, Fim-2/c-fms, and Fim-3, three common integration sites of Friend murine leukemia virus in myeloblastic leukemias, map to mouse chromosomes 13, 18, and 3, respectively
Authors: Sola B , Simon D , Mattéi M G , Fichelson S , Bordereaux D , Tambourin P E , Guenet J L , Gisselbrecht S
Published: January 2020

Three common proviral integration sites, Fim-1, Fim-2/c-fms, and Fim-3, have been described in mouse myeloid leukemias induced by the Friend murine leukemia virus. The nature and function of Fim-1 and Fim-3 are still unknown since no transcript from these loci has been detected so far. To identify these two loci, we undertook their chromosomal localization using restriction fragment length polymorphism detected between C57BL/6 mice and the wild-derived inbred strain of Mus spretus. Using interspecific backcross analysis, we mapped Fim-1 to mouse chromosome 13 and Fim-3 to mouse chromosome 3. Interestingly, Fim-3 is tightly linked to Evi-1, another common integration site of ecotropic virus involved in another model of mouse myeloid leukemogenesis. Fim-2 spans the 5' end of the c-fms gene, which encodes for the macrophage-colony-stimulating factor receptor. We located the c-fms gene on the D band of chromosome 18 by in situ hybridization.
Synthesis and processing of the Marek's disease herpesvirus B antigen glycoprotein complex
Authors: Sithole I , Lee L F , Velicer L F
Published: January 2020

The Marek's disease herpesvirus B antigen (MDHV-B) complex was previously immunologically identified and molecularly characterized as a set of three glycoproteins designated gp100, gp60, and gp49 on the basis of apparent molecular weight and immunoprecipitation with both polyclonal and monoclonal antibodies. Immunoprecipitation analysis, previously with polyclonal and more recently with monoclonal antibodies, of infected cell lysates labeled with [35S]methionine in the presence of tunicamycin, an inhibitor of N-linked glycosylation, revealed two putative precursor molecules of 88,000 daltons (pr88) and 44,000 daltons (pr44). High-resolution pulse-chase studies revealed that gp100 was a glycosylated intermediate which was processed to yield gp60 and gp49. This cleavage was inhibited by monensin, an inhibitor of glycoprotein processing. Endo-beta-N-acetylglucosaminidases F and H (endo-F, endo-H) reduced gp100 to pr88, indicating that the latter is an intermediate in the biosynthetic pathway. These same enzymes reduced gp49, and to a lesser extent gp60, to pr44, suggesting that pr44 is their polypeptide backbone. Significant support for this concept is the fact that the same monoclonal antibody recognized all three molecules, gp60, gp49, and pr44. In the presence of monensin, terminal addition of complex sugars was also prevented, since gp60 was replaced by a slightly faster migrating component which was insensitive to both endo-F and endo-H. Cell-free translation of infected-cell mRNA, followed by immunoprecipitation analysis with either polyclonal or monoclonal antibody, resulted in detection of a putative unglycosylated precursor polypeptide of 44,000 daltons. Since pr88 was not the initial precursor polypeptide of the MDHV-B complex, its existence may have resulted from dimerization of pr44. Again, detection of both pr88 and pr44 with the same monoclonal antibody is consistent with this interpretation. These collective data obtained from the cell-free and in vivo studies with polyclonal and monoclonal antibodies reactive with MDHV-B are consistent with the concept that pr44, the initial gene product, dimerizes to form pr88 and demonstrate that pr88 is actually a processing intermediate glycosylated to gp100, another processing intermediate, which is then processed to gp60 and gp49.
Different mechanisms account for the relative resistance of KG-1 and HL-60 cell lines to retrovirus infection
Authors: Collins S J
Published: January 2020

I infected three different human leukemic cell lines (K562, KG-1, and HL-60) with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. Compared with K562 cells, both KG-1 and HL-60 cells were relatively resistant to infection with this retrovirus vector. In HL-60 cells, this resistance appeared to result from diminished viral DNA synthesis, while in KG-1 cells there was a block to the genomic integration of the viral DNA.
Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells
Authors: Holmes A M , Wietstock S M , Ruyechan W T
Published: January 2020

A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. In contrast, the pH optimum of the HeLa DNA primase was very sharp and fell between pH 7.9 and 8.2. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase and was eluted from single-stranded DNA agarose at higher salt concentrations than the host primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degrees C for several weeks, the DNA primase separated from the viral DNA polymerase. Separation or decoupling could also be achieved by gel filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA.
Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide
Authors: Jagadish M N , Staton V J , Hudson P J , Azad A A
Published: January 2020

The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.
Identification and characterization of a varicella-zoster virus DNA-binding protein by using antisera directed against a predicted synthetic oligopeptide
Authors: Kinchington P R , Inchauspe G , Subak-Sharpe J H , Robey F , Hay J , Ruyechan W T
Published: January 2020

We have identified, in varicella-zoster virus (VZV)-infected cells, the product of the gene predicted to code for the VZV analog of the herpes simplex virus major DNA-binding protein. The open reading frame of the VZV gene has the potential to code for a protein with a predicted molecular weight of 132,000 (a 132K protein). To detect the protein, a 12-amino-acid oligopeptide corresponding to the carboxyl terminus of the putative open reading frame was synthesized and used to prepare antisera in rabbits. The resulting antibodies reacted specifically in Western immunoblot analysis and immunoprecipitation with a single 130K polypeptide found in VZV-infected cells. The specific reactivity of the antisera with the 130K polypeptide was inhibited by the addition of synthetic peptide. Immunofluorescence studies with the antisera as probe for the 130K polypeptide suggested that this peptide is located predominantly within the nuclei of infected cells. Analysis of proteins that bind to single-stranded DNA immobilized on cellulose matrices indicated that 30 to 50% of the 130K polypeptide is capable of interacting with single-stranded DNA and that this interaction is overcome with 0.5 M NaCl. Thus, we have prepared a specific polyclonal antiserum that identifies a VZV DNA-binding protein whose properties are similar to those of the herpes simplex virus ICP8 (Vmw130) DNA-binding protein.
Biological activities of oligonucleotides spanning the F9 point mutation within the enhancer region of polyomavirus DNA
Authors: Satake M , Furukawa K , Ito Y
Published: January 2020

A mutant of polyomavirus, F441, selected to grow in undifferentiated mouse F9 embryonal carcinoma cells, carries a single-base change in the enhancer region at nucleotide (nt) 5233 of the viral genome. Enhancers of most of the F9 mutants have a duplicated segment of viral DNA encompassing nt 5233. The minimum duplicated segment of all the known F9 mutants is from nt 5218 to nt 5239. We prepared oligonucleotides spanning the sequence from nt 5218 through nt 5239 of the genome of the wild type and F441 and examined the biological activities of the oligonucleotides by a transient assay of chloramphenicol acetyltransferase (CAT) gene expression in F9 cells. The oligonucleotide harboring the F441 mutation was shown to increase cat gene expression in F9 cells when linked at an upstream position in both orientations. When dimerized at an upstream position, the F441 oligonucleotide showed even higher cat gene expression enhancing activity. In contrast, no such effects were observed with the oligonucleotide of the wild-type sequence. In addition, the F441 oligonucleotide, but not the wild-type sequence, could inhibit the activity of whole enhancer fragment of F441 when cotransfected into F9 cells in excess amounts. On the basis of the results obtained, we suggest that the segment of F441 enhancer encompassing the point mutation contains a target for a cellular factor(s) which acts in a positive manner to increase the transcription of a gene in undifferentiated mouse F9 cells.
Mechanism of selection of class II recombinant murine leukemia viruses in the highly leukemic strain CWD
Authors: Thomas C Y , Roberts J S , Buxton V K
Published: January 2020

The development of spontaneous lymphomas in CWD mice is associated with the expression of endogenous ecotropic murine leukemia viruses (MuLV) and the formation of recombinant viruses. However, the pattern of substitution of nonecotropic sequences within the envelope genes of the CWD class II recombinant viruses differs from that seen in class I recombinant MuLVs of AKR, C58, and HRS mice. To determine how CWD host genes might influence the envelope gene structure of the recombinant viruses, we characterized the responses of these mice to two different types of exogenous MuLVs. Neonatal mice injected the HRS class I recombinant PTV-1 became infected and developed T-cell lymphomas more rapidly than controls did. The inoculation of CWD mice with the leukemogenic AKR ecotropic virus SL3-3 led to the formation of recombinant MuLVs with a novel genetic structure and class II-like envelope genes, although SL3-3 generates class I recombinants in other strains. These results suggest that the absence of class I recombinant MuLVs in CWD mice is not related to the restriction of the replication or oncogenicity of class I viruses or to the absence of an appropriate ecotropic virus that can generate class I recombinants. More likely, the genes of CWD mice that direct the formation or selection of class II recombinant viruses affect the process of recombination between the ecotropic and nonecotropic envelope gene sequences.
Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI
Authors: Johnson D C , Frame M C , Ligas M W , Cross A M , Stow N D
Published: January 2020

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.
Influenza virus infection of tracheal gland cells in culture
Authors: Gentry S E , Culp D J , Roberts N J , Marin M G , Simons R L , Latchney L R
Published: January 2020

Influenza virus-induced tracheobronchitis causes limited epithelial deciliation but markedly decreased mucociliary transport. This suggests that virus-induced alterations in airway mucus play a role in decreased mucociliary transport. Airway submucosal glands are a primary source of mucus. Therefore, we examined virus-gland cell interactions by exposing primary cultures of isolated feline tracheal gland cells to influenza A/Scotland/840/74 H3N2 virus for 1 h at a multiplicity of infection of 0.1. Virus production and release into the culture medium first occurred between 8 and 12 h postinfection and eventually reached a steady state that continued for at least 8 days. Virus which was produced and released by infected cells infected other monolayers, resulting in viral production similar to that after infection with stock virus. Hemadsorption assays conducted 24 h after infection demonstrated that most of the cells in a monolayer became infected. The infection was nonlytic according to cell morphology, trypan blue dye exclusion, and release of lactate dehydrogenase. Because lysis of a cell subpopulation could have been masked by subsequent cell division, we compared the uptake of [3H]thymidine by infected and control monolayers. There was no increase in uptake by infected monolayers. These results demonstrate that feline tracheal gland cells in primary culture undergo productive and nonlytic infection with influenza A virus. This model provides a unique system for the study of virus-gland interactions isolated from the influence of other tissues.
Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins
Authors: Martin S , Courtney R J , Fowler G , Rouse B T
Published: January 2020

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.
Purification of the herpes simplex virus type 1 65-kilodalton DNA-binding protein: properties of the protein and evidence of its association with the virus-encoded DNA polymerase
Authors: Gallo M L , Jackwood D H , Murphy M , Marsden H S , Parris D S
Published: January 2020

Using a combination of conventional column chromatography and velocity sedimentation, we have purified the 65-kilodalton DNA-binding protein (65KDBP) encoded by herpes simplex virus (HSV) greater than 625-fold. The HSV type 1 (HSV-1)-encoded DNA polymerase (pol) cofractionated with 65KDBP through DEAE-Sephacel, Blue Sepharose, and Mono Q columns and was only separated from 65KDBP by sedimentation through a glycerol gradient. Immunoaffinity columns containing monoclonal antibody (MAb) 6898 immunoglobulin effectively bound most of the HSV-1 pol activity which coeluted with 65KDBP. The pattern of reactivities of HSV-1/HSV-2 recombinants with MAbs specific for HSV-1 65KDBP or the HSV-2-infected cell-specific protein ICSP34,35 strongly suggests that these two species are serotype equivalents of the same protein. Taken together, all these data indicate that 65KDBP is a pol-associated protein and the HSV-1 counterpart of HSV-2 ICSP34,35 previously reported to have similar properties (P. J. Vaughan, D. J. M. Purifoy, and K. L. Powell, J. Virol. 53:501-508, 1985). Purified preparations of 65KDBP were capable of binding to double-stranded DNA, as determined by filter retention and mobility shift assays. The protein-DNA complex formed with 65KDBP was distinct from that produced by pol and could be further shifted by the addition of immunoglobulin specific for 65KDBP. These results demonstrate that 65KDBP has been purified substantially free from pol and indicate that DNA binding is an inherent property of the protein.
Genetic analysis of in vivo-selected viral variants causing chronic infection: importance of mutation in the L RNA segment of lymphocytic choriomeningitis virus
Authors: Ahmed R , Simon R S , Matloubian M , Kolhekar S R , Southern P J , Freedman D M
Published: January 2020

Viral variants with different biological properties are present in the central nervous systems (CNS) and lymphoid tissues of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Viral isolates from the CNS are similar to the original Armstrong LCMV strain and induce potent virus-specific T-cell responses in adult mice, and the infection is rapidly cleared. In contrast, LCMV isolates derived from spleens of carrier mice cause persistent infections in adult mice. This chronic infection is associated with low levels of antiviral T-cell responses. In this study, we genetically characterized two independently derived spleen variants by making recombinants (reassortants) between the spleen isolates and wild-type (wt) LCMV and showed that the ability to persist in adult mice and the associated suppression of T-cell responses segregates with the large (L) RNA segment. In addition, we analyzed a revertant (isolated from the CNS) derived from one of the spleen variants. By comparing the biological properties of three reassortants that contained the same S segment but had the L segment of either the original wt Armstrong LCMV, the spleen variant derived from it, or the CNS revertant derived from the spleen variant, we were able to show unequivocally that biologically relevant mutations occurred in the L segment not only during generation of the spleen variant from wt LCMV but also in reversion of the spleen variant to the wt phenotype. Thus, our results showed that (i) genetic alterations in the L genomic segment were involved in organ-specific selection of viral variants, and (ii) these mutations profoundly affected the ability of LCMV to cause chronic infections in adult mice.
Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus
Authors: Smith R F , Smith T F
Published: January 2020

By using amino acid sequence patterns (motifs) diagnostic of conserved regions within the catalytic domains of protein kinases, homologous open reading frames of three herpesviruses were identified as protein kinase-related genes. The three sequences, herpes simplex virus gene UL13, varicella-zoster virus gene 47, and Epstein-Barr virus gene BGLF4, resemble serine/threonine kinases rather than tyrosine kinases.
Trojan horse lymphocytes: a vesicular stomatitis virus-specific T-cell clone lyses target cells by carrying virus
Authors: Hom R C , Soman G , Finberg R
Published: January 2020

We have isolated a vesicular stomatitis virus (VSV)-specific CD4+ CD8- murine T-cell clone. This clone proliferates only in response to VSV and lyses infected tumor cells bearing class II major histocompatibility antigens in short-term chromium release assays. In addition, the cell has VSV antigens on its surface and is capable of killing uninfected tumor cells without major histocompatibility antigen restriction in a 2-day assay. This latter cytolytic activity is eliminated by anti-VSV antibody, indicating that its lytic activity is provided by the virus. [35S]methionine labeling and immunoprecipitation experiments demonstrated that viral protein translation is initiated after incubation of the clone with a tumor target cell, defining this as the mechanism of its cytolytic activity.
Single-amino-acid substitution in an antigenic site of influenza virus hemagglutinin can alter the specificity of binding to cell membrane-associated gangliosides
Authors: Suzuki Y , Kato H , Naeve C W , Webster R G
Published: January 2020

Antigenic variants of influenza virus A/Mem/1/71-Bel/42 (H3N1) selected with monoclonal antibodies and having single substitutions in their hemagglutinins were examined for their ability to hemagglutinate and hemolyse erythrocytes coated with different gangliosides. The majority of variants, including one with a substitution near the receptor-binding site (Asn-133----Lys), did not differ from the parent in specificity for receptor molecules. However, a substitution in HA1 at residue 205 (Ser----Tyr), which is distant from the receptor-binding site in antigenic site D, affected hemagglutination and hemolysis of erythrocytes coated with sialyl-paraglobosides. The variant preferentially recognized N-acetylneuraminic acid-alpha 2,6-galactose linkages to sialylparaglobosides, whereas the parent and other variants preferentially recognized N-acetylneuraminic acid-alpha 2,3-galactose linkages. In the trimeric hemagglutinin molecule, residue 205 is located across the subunit interface from the receptor-binding site. The bulky hydrophobic tyrosine in the variant may cause a conformational change in the receptor-binding pocket on the neighboring subunit and influence receptor binding.
Differential DNA binding of nuclear proteins to a long terminal repeat region of the MCF13 and Akv murine leukemia viruses
Authors: Yoshimura F K , Tupper J , Diem K
Published: January 2020

Long terminal repeat (LTR) sequences of murine leukemia viruses (MLVs) have been demonstrated to be mainly responsible for the pathogenic differences in these retroviruses. A region of the LTR which is downstream of the enhancer elements has been shown to contribute both to enhancer activity as well as to disease specificity of MLVs. We have identified protein-DNA complexes generated by this region of a lymphomagenic MLV (MCF13) and one which is nonpathogenic (Akv). One protein-DNA complex we have observed for this region is unique to MCF13 DNA sequences. Detection of protein involved in this unique MCF13 complex in different cell lines revealed that it was ubiquitous.
Expression of the int-1 and int-2 loci in endogenous mouse mammary tumor virus-induced mammary tumorigenesis in the C3Hf mouse
Authors: Etkind P R
Published: January 2020

The int-1 locus appears to be involved in over 80% of C3H exogenous mouse mammary tumor virus (MMTV)-induced mouse mammary tumors, and the int-2 locus appears to be involved in approximately 10% of these tumors. Analysis of 46 C3Hf mammary tumors resulting from endogenous, rather than exogenous, MMTV infection revealed that only 41% expressed int-1 RNA, while 2% expressed int-2 RNA. Our results suggest that in addition to the int-1 and int-2 loci, other loci may be involved in endogenous-MMTV-induced mammary tumors of the C3Hf mouse.
Arrangement of repetitive sequences in the genome of herpesvirus Sylvilagus
Authors: Medveczky M M , Geck P , Clarke C , Byrnes J , Sullivan J L , Medveczky P G
Published: January 2020

Herpesvirus sylvilagus is a lymphotropic (type gamma) herpesvirus of cottontail rabbits (Sylvilagus floridanus). Analysis of virion DNA of herpesvirus sylvilagus has revealed that the genome consists of one stretch of about 120 kilobase pairs of internal, unique DNA flanked by a variable number of 553-base-pair tandem repeats. The G + C content of the repetitive DNA is extremely high (83%), as determined by sequencing. The organization of the herpesvirus sylvilagus genome is, therefore, similar to that of the primate lymphotropic viruses herpesvirus saimiri and herpesvirus ateles.
Regulation of poly(A) site selection in adenovirus
Authors: Falck-Pedersen E , Logan J
Published: January 2020

We have investigated the mechanisms involved in the early-to-late RNA-processing switch which regulates the mRNA species generated from the adenovirus major late transcription unit (MLTU). In particular, polyadenylation choice mechanisms were characterized by using a reconstructed adenovirus E1A gene as a site for insertion of MLTU poly(A) regulation signals (L1 and L3). Adenovirus constructs containing the variant poly(A) recognition elements were used to compare E1A poly(A) signal utilization with wild-type MLTU (L1 to L5) utilization. In both early and late stages of infection, either polyadenylation site (L1 or L3) is capable of being utilized when presented as the only operational poly(A) site. In an early infection, a virus which contains multiple elements presented in tandem (L13) uses the first poly(A) site, L1, preferentially (ratio of L1 to L3, 8:1) in both E1A and MLTU loci. Transcription termination is not involved in restricting the utilization of the downstream L3 site. In a late infection, when each of the five MLTU poly(A) sites is used, a switch also occurs for the E1AL13 construct, with utilization of both the L1 and L3 poly(A) sites. The switch from early to late was not the result of altered processing factors in the late infection, as demonstrated by superinfecting the E1AL13 construct into cells which had already entered a late stage of infection. The superinfecting virus gave an L1-only phenotype; therefore, a cis mechanism is involved in adenovirus poly(A) regulation.
Acquisition of new proviral copies in avian lymphoid cells transformed by reticuloendotheliosis virus
Authors: Zhang J Y , Bose H R
Published: January 2020

The expression of the v-rel oncogene of avian reticuloendotheliosis virus (REV-T) transforms and immortalizes very immature avian lymphoid cells. In REV-T-transformed lymphoid cells which were persistently infected with reticuloendotheliosis-associated virus (REV-A), the REV-T proviral copy number increases after the initial integration event. In 23 independently derived REV-T-transformed cell lines, 15 of the 18 virus-producing cell lines had acquired additional proviruses. The rate at which the newly acquired proviral sequences accumulated differed for various cell lines. In some cell lines, additional REV-T proviral copies could be detected as early as 8 months after the initial integration event. A correlation exists between the number of REV-T proviral sequences and the length of time which a given cell line had been propagated in culture. The integration sites occupied by the newly acquired REV-T proviruses were distinct. In contrast, reticuloendotheliosis-associated virus proviral sequences in these REV-T-transformed virus-producing lymphoid cells did not increase during in vitro culture. Furthermore, the acquisition of additional REV-T proviral sequences did not occur in non-virus-producing cell lines. Two of the newly acquired proviral sequences were molecularly cloned and analyzed by restriction endonuclease mapping. Although the newly acquired REV-T proviruses have not sustained major deletions, the viral sequences and the v-rel oncogene display numerous restriction enzyme polymorphisms. The cellular flanking sequences of two newly acquired REV-T proviruses analyzed were unique and shared no homology with flanking sequences of the other REV-T proviruses in these transformed cells. The nucleotide sequence of the virus-cellular DNA junctions of one newly acquired provirus and its cellular sequence prior to proviral integration were defined. A 5-base-pair direct repeat of cellular origin was present on each side of the long terminal repeat, indicating that the mechanism of acquisition of additional REV-T proviral sequences used reverse transcription and integration of new REV-T proviral copies.
Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters
Authors: Cromlish W A , Abmayr S M , Workman J L , Horikoshi M , Roeder R G
Published: January 2020

In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements.
Detection of virus-specific RNA-dependent RNA polymerase activity in extracts from cells infected with lymphocytic choriomeningitis virus: in vitro synthesis of full-length viral RNA species
Authors: Fuller-Pace F V , Southern P J
Published: January 2020

We have developed an in vitro assay for the lymphocytic choriomeningitis virus (LCMV) RNA-dependent RNA polymerase with ribonucleoprotein complexes extracted from acutely infected tissue culture cells. The RNA products synthesized in vitro corresponded in size to the full-length genomic L and S RNAs and subgenomic NP and GP mRNAs normally produced in vivo during acute LCMV infection. In a temporal analysis spanning the first 72 h of acute infection, the in vitro polymerase activity of ribonucleoprotein complexes was maximal at 16 h and declined significantly at later times. In contrast, the intracellular levels of the viral L protein (the putative polymerase protein) appeared to be maximal at 48 to 72 h postinfection. Our results suggest that the accumulation of L protein correlates with reduced viral replication and transcription at later times in acute infection and may be involved in the transition from acute to persistent LCMV infection.
Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region
Authors: Pontisso P , Petit M A , Bankowski M J , Peeples M E
Published: January 2020

Hepatitis B virus particles contain three related viral envelope proteins, the small, middle, and large S (surface) proteins. All three proteins contain the small S amino acid sequence at their carboxyl terminus. It is not clear which of these S proteins functions as the viral attachment protein, binding to a target cell receptor and initiating infection. In this report, recombinant hepatitis B surface antigen (rHBsAg) particles, which contain only virus envelope proteins, were radioactively labeled, and their attachment to human liver membranes was examined. Only the rHBsAg particles containing the large S protein were capable of directly attaching to liver plasma membranes. The attachment was saturable and could be prevented by competition with unlabeled particles or by a monoclonal antibody specific for the large S protein. In the presence of polymerized human serum albumin, both large and middle S protein-containing rHBsAg particles were capable of attaching to the liver plasma membranes. Small S protein-containing rHBsAg particles were not able to attach even in the presence of polymerized human serum albumin. These results indicate that the large S protein may be the viral attachment protein for hepatocytes, binding directly to liver plasma membranes by its unique amino-terminal (pre-S1) sequence. These results also indicate that polymerized human serum albumin or a similar molecule could act as an intermediate receptor, attaching to liver plasma membranes and to the amino acid sequence (pre-S2) shared by the middle and large S proteins but not contained in the small S protein.
Dengue virus-specific murine T-lymphocyte proliferation: serotype specificity and response to recombinant viral proteins
Authors: Rothman A L , Kurane I , Zhang Y M , Lai C J , Ennis F A
Published: January 2020

Definition of the T-lymphocyte responses to dengue viruses should aid in the development of safe and effective vaccines and help to explain the pathophysiology of dengue hemorrhagic fever and dengue shock syndrome. In this study, we demonstrated that dengue virus-specific T lymphocytes were detected in spleen cells from dengue virus-immune mice using an in vitro proliferation assay. Following immunization with a single dose of infectious dengue virus, murine lymphocytes showed increased proliferation when incubated in the presence of viral antigens of the same serotype but not in the presence of control antigens. Depletion experiments with antibody and complement showed that the population of responding cells expressed the Thy1+ L3T4+ Lyt2- phenotype. This indicates that the predominant proliferating cells are T lymphocytes of the helper-inducer phenotype. Dengue virus-specific memory lymphocyte responses were detectable for at least 22 weeks after immunization. The response to primary infection was primarily serotype specific, with some serotype cross-reactivity present at a low level. We demonstrated that lymphocytes from mice immunized with dengue 4 virus proliferate in response to a combination of dengue 4 virus C, pre-M, E, NS1, and NS2a proteins expressed in Sf9 cells with a recombinant baculovirus, and, to a lesser extent, to the dengue 4 virus E protein alone.
Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus
Authors: Ffrench R A , Tang X L , Anders E M , Jackson D C , White D O , Drummer H , Wade J D , Tregear G W , Brown L E
Published: January 2020

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.