bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of an endogenous retrovirus enhancer by insertion into a heterologous context
Authors: Conklin K F
Published: January 2020

An enhancer element is located in the U3 portion of exogenous avian retrovirus long terminal repeats (LTRs). A similar element has not been detected in the LTRs of ev-1 and ev-2, two avian endogenous viruses (evs) that normally are not expressed in vivo. Experiments were initiated to determine whether minor nucleotide differences in the U3 region of a previously untested ev that is ubiquitously expressed in vivo (ev-3) might confer enhancer function on the LTR of this provirus. This question was addressed by inserting U3 regions from ev-3 and from ev-1 and/or ev-2 both upstream of the herpesvirus thymidine kinase gene promoter and in place of the major enhancer domains of the Rous sarcoma virus LTR and determining their relative effects on transcription. U3 regions from all evs tested were unable to enhance transcription from the thymidine kinase gene promoter, indicating that nucleotide differences in the ev U3 regions do not affect their relative enhancer function and therefore are unlikely to play a role in their differential expression in vivo. Unexpectedly, however, all ev U3 regions were able to augment transcription in an orientation-independent manner in the ev-Rous sarcoma virus hybrid LTRs. Further experiments conducted to determine why this enhancer activity is not detectable in intact ev LTRs demonstrated that it was not due to removal of repressor sequences in the ev fragments used that might normally be present in intact ev LTRs. The lack of detectable enhancer activity in intact ev LTRs also was not explained by a defect in ev promoters that makes them unresponsive to enhancers in cis. These experiments therefore identify sequences that, although unable to function detectably as enhancers in their natural context, can function efficiently in a heterologous context. Data are discussed in terms of the modularity of enhancer elements and possible interactions between enhancers and promoter-specific sequences.
Age-dependent resistance to murine retrovirus-induced spongiform neurodegeneration results from central nervous system-specific restriction of virus replication
Authors: Czub M , Czub S , McAtee F J , Portis J L
Published: January 2020

The murine retrovirus CasBrE causes a noninflammatory spongiform degeneration of the central nervous system (CNS). Mice inoculated as neonates develop viremia and are susceptible to disease. However, mice inoculated at 10 days of age do not develop viremia and are totally resistant to the neurologic disease. We recently described a highly neurovirulent chimeric virus, FrCasE (J. L. Portis, S. Czub, C. F. Garon, and F. J. McAtee, J. Virol. 64:1648-1656, 1990), which contains the env gene of CasBrE. Mice inoculated at 10 days of age with this virus developed a viremia comparable to that in neonatally inoculated mice but, surprisingly, were still completely resistant to the neurodegenerative disease. A comparison of the tissue distribution of virus replication for mice inoculated at 1 or 10 days of age was determined by Southern blot analysis for the quantification of viral DNA and by infectious-center assay for the quantification of virus-producing cells. The levels of virus replication in the spleens were comparable in the two groups. In contrast, virus replication in the CNS of the resistant 10-day-old mice was markedly restricted (100- to 1,000-fold). Intracerebral inoculation did not overcome this restriction. A similar pattern of CNS-specific restriction of virus replication and resistance to disease was observed in athymic NIH Swiss nude mice inoculated at 10 days of age, suggesting that T-cell immunity was not involved. From our results, we conclude that the age-dependent resistance to disease is a consequence of the restriction of virus replication within the CNS due to the developmental state of the organ.
Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation
Authors: Cohen J I , Wang F , Kieff E
Published: January 2020

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.
Transdominant human T-cell lymphotropic virus type I TAX1 mutant that fails to localize to the nucleus
Authors: Gitlin S D , Lindholm P F , Marriott S J , Brady J N
Published: January 2020

Human T-cell lymphotropic virus type I (HTLV-I) encodes a 40-kDa nuclear transactivating phosphoprotein, TAX1. The results presented in this study demonstrate that deletion of amino acids 2 through 59 of TAX1 (delta 58 TAX1) decreased transactivation of the HTLV-I long terminal repeat 10- to 20-fold. S1 nuclease analysis revealed that the decrease in transactivation of the HTLV-I long terminal repeat was associated with a lack of RNA synthesis. In contrast to the nuclear localization of the wild-type TAX1 protein, indirect immunofluorescence analysis demonstrated that delta 58 TAX1 failed to localize to the nucleus, indicating that the TAX1 nuclear localization sequence is present in amino acids 2 through 59. Cotransfection of wild-type and mutant TAX1 DNAs resulted in the cytoplasmic accumulation of TAX1 and a 25-fold decrease in transactivation. Although several possibilities which may account for this transdominant effect exist, we favor a model in which delta 58 TAX1 interferes with the nuclear localization of wild-type TAX1 protein, perhaps by forming heterodimer complexes.
Mutations in the regions of the Rous sarcoma virus 3' splice sites: implications for regulation of alternative splicing
Authors: Berberich S L , Stoltzfus C M
Published: January 2020

Retrovirus RNA is synthesized as a single primary transcript that is differentially processed by RNA splicing. Three species of viral RNA (spliced env, spliced src, and unspliced full-length RNA) are produced in chicken embryo fibroblasts infected with Rous sarcoma virus, an avian retrovirus. The env and src mRNAs are synthesized by the alternative use of two 3' splice sites. The mechanism by which balanced splicing at the two sites is maintained was investigated in this report. Mutants that increase or decrease splicing at one of the two 3' splice sites were analyzed for the effect on splicing at the other site. The two splice sites differed in their response. Mutations that caused a specific increase in the level of spliced env mRNA were associated with reciprocal changes in the levels of src mRNA but with no change in overall splicing. In contrast, mutants in which the src 3' splice site was inactivated demonstrated a decreased overall level of spliced RNA but had little or no effect on the level of spliced env mRNA. Mutations that caused specific increases in the level of spliced src mRNA had variable effects on env mRNA levels. Deletion of regions in gag, which was previously shown to contain a cis-acting negative regulator of splicing, resulted in a corresponding increase of both spliced viral mRNAs and a decrease in unspliced RNA, suggesting that this element suppressed both env and src splicing. Several models are discussed which are possible mechanisms for regulation of alternative splicing of Rous sarcoma virus RNA, but none of these models appear to be consistent with all of the data.
Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression
Authors: Besansky N J , Butera S T , Sinha S , Folks T M
Published: January 2020

Using a polymerase chain reaction-based assay on total cell lysates, we have detected unintegrated human immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4 inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction. ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, J1 cells, with surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.
High-efficiency formation of influenza virus transfectants
Authors: Enami M , Palese P
Published: January 2020

cDNA-derived RNAs were introduced into the genomes of influenza viruses by using an improved ribonucleoprotein (RNP) transfection protocol. Up to 10(5) viral transfectants with a novel neuraminidase gene could be obtained by using a 35-mm dish (10(6) cells) for RNP transfection. In addition to genes coding for surface proteins (hemagglutinin and neuraminidase), we also exchanged a gene coding for nonsurface proteins. The cDNA-derived influenza A/PR/8/34 virus NS gene was introduced into a temperature-sensitive mutant with a defect in this gene. We suggest that the term influenza virus transfectant be used for those viruses which are made by RNP transfection with cDNA-derived RNA.
Transactivation of the human immunodeficiency virus promoter by human herpesvirus 6 (HHV-6) strains GS and Z-29 in primary human T lymphocytes and identification of transactivating HHV-6(GS) gene fragments
Authors: Horvat R T , Wood C , Josephs S F , Balachandran N
Published: January 2020

Human herpesvirus 6 (HHV-6) can activate the human immunodeficiency virus (HIV) promoter and accelerate cytopathic effects in HIV-infected human T cells. This study examines the regions of the HIV promoter required for HHV-6 transactivation in a heterogeneous population of primary human T lymphocytes with or without antigenic stimulation. Two different strains of HHV-6, GS and Z29, transactivated the HIV promoter. The GS strain transactivated the promoter in both stimulated and resting T cells, while the Z29 strain increased HIV promoter activity only in stimulated T cells. Three DNA clones containing HHV-6(GS) genomic fragments transactivated the HIV promoter in cotransfected T cells. A 21.4-kb DNA clone, pZVB70, showed the highest transactivating ability, while two other DNA fragments, pZVB10 (6.2 kb) and pZVH14 (8.7 kb), showed lower activity. One of these clones, pZVH14, activated the HIV promoter construct containing a mutation in the NF kappa B site. However, this mutated NF kappa B promoter was not transactivated during HHV-6(GS) infection or after cotransfection with pZVB70 or pZVB10. These data indicate that the NF kappa B sites of the HIV promoter are essential for its transactivation during HHV-6(GS) infection. By increasing HIV promoter activity in primary T lymphocytes, HHV-6 may consequently increase HIV replication, leading to an increase in the cytopathic effect on coinfected human T cells.
Identification of the simian foamy virus transcriptional transactivator gene (taf)
Authors: Mergia A , Shaw K E , Pratt-Lowe E , Barry P A , Luciw P A
Published: January 2020

Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).
Origin of adeno-associated virus DNA replication is a target of carcinogen-inducible DNA amplification
Authors: Yalkinoglu A O , Zentgraf H , Hübscher U
Published: January 2020

DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semi-conservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount of DpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase alpha, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.
Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27
Authors: Banks T A , Allen E M , Dasgupta S , Sandri-Goldin R , Rouse B T
Published: January 2020

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.
Very high incidence of germ cell tumorigenesis (seminomagenesis) in human papillomavirus type 16 transgenic mice
Authors: Kondoh G , Murata Y , Aozasa K , Yutsudo M , Hakura A
Published: January 2020

Human papillomavirus type 16 (HPV16) is frequently found in carcinomas and precancerous lesions of the uterine cervix and is thought to be closely associated with carcinogenesis in these regions. However, the transforming activity of the E6 and E7 genes in vivo has not been characterized. To investigate this function, we produced transgenic mice carrying HPV16 E6 and E7 open reading frames. We obtained five transgenic founders and established three transgenic lineages. We observed testicular tumors of germ cell origin in mice of all three lineages. Morphological studies showed that these tumors were a type of seminoma. Both testes of all tumor-bearing mice were affected with this type of tumor. Strikingly, in one lineage, all of the male mice developed this tumor. On Northern (RNA) analysis, a high level of expression of HPV mRNA was detected in these tumors. These results suggest that transforming genes of HPV16 have transforming activity in vivo and preferential effects on germ cells in the testis.
A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release
Authors: Schmutz C , Payne L G , Gubser J , Wittek R
Published: January 2020

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.
Bovine papillomavirus type 1 alters the processing of host glucose- and calcium-modulated endoplasmic reticulum proteins
Authors: O'Banion M K , Young D A
Published: January 2020

We have previously characterized five proteins induced by the presence of the E2 open reading frame (ORF) region of bovine papillomavirus type 1 (BPV-1) in C127 mouse fibroblasts (R. M. Levenson, U. G. Brinckmann, M. K. O'Banion, E. J. Androphy, J. T. Schiller, F. Tabatabai, L. P. Turek, K. Neary, M. T. Chin, T. R. Broker, L. T. Chow, and D. A. Young, Virology 172:170-179, 1989). By specific immunoprecipitation, we now find that one of the papillomavirus-associated proteins (pvp1) is a highly glycosylated form of glucose-regulated protein 100 (grp100), a major constituent of the endoplasmic reticulum. A second set of pvps (2, 3, and 4) are shown to be related precursors of another protein already present in C127 cells (protein B). Based on their induction by the calcium ionophore A23187 and their positions on giant two-dimensional gels, we have tentatively identified pvp2, -3, and -4 and B as forms of calcium-regulated protein 55, another constituent of the endoplasmic reticulum (D. R. J. Macer and G. L. E. Koch, J. Cell Sci. 91:61-70, 1988). The mechanism by which BPV-1 brings about these changes is not yet defined; however, it is unlikely to involve calcium level perturbations or transformation per se, since ionophore treatment changes other proteins in C127 cells not seen with BPV and the papillomavirus-associated proteins are found in nontransformed cells harboring the E2 ORF region. Furthermore, the BPV changes are not associated with increased grp mRNA levels, as occurs in ionophore-treated cells. Rather, it appears that BPV-1 somehow retards the normal processing of these resident endoplasmic reticulum proteins that are believed to serve as critical regulators of host protein processing and assembly.
Role of alpha-transinducing factor (VP16) in the induction of alpha genes within the context of viral genomes
Authors: Spector D , Purves F , Roizman B
Published: January 2020

In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.
Flux of the paramyxovirus hemagglutinin-neuraminidase glycoprotein through the endoplasmic reticulum activates transcription of the GRP78-BiP gene
Authors: Watowich S S , Morimoto R I , Lamb R A
Published: January 2020

The cellular glucose-regulated protein GRP78-BiP is a member of the HSP70 stress family of gene products, and the protein is a resident component of the endoplasmic reticulum, where it is thought to play a role in the folding and oligomerization of secretory and membrane-bound proteins. GRP78-BiP also binds to malfolded proteins, and this may be one mechanism for preventing their intracellular transport. An induction in synthesis of the GRP78-BiP protein occurs in cells infected with paramyxoviruses (R. W. Peluso, R. A. Lamb, and P. W. Choppin, Proc. Natl. Acad. Sci. USA 75:6120-6124, 1978). We have studied the expression and activity of the GRP78-BiP gene and synthesis of the GRP78-BiP protein during infection with the paramyxovirus simian virus 5 (SV5). We wished to identify the viral component capable of causing activation of GRP78-BiP since GRP78-BiP interacts specifically and transiently with the SV5 hemagglutinin-neuraminidase (HN) glycoprotein during HN folding (D. T. W. Ng, R. E. Randall, and R. A. Lamb, J. Cell Biol. 109:3273-3289, 1989). Expression of cDNAs of the SV5 wild-type HN glycoprotein and a mutant form of HN that is malfolded but not the SV5 F glycoprotein or SV5 cytoplasmic proteins P, V, and M caused increased amounts of GRP78-BiP mRNA to accumulate, as detected by nuclease S1 protection assays. As unfolded or malfolded forms of HN cannot be detected to accumulate during SV5 infection, the data suggest that the flux of HN through the ER in SV5-infected cells can cause activation of GRP78-BiP transcription.
Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope
Authors: Del Val M , Schlicht H J , Volkmer H , Messerle M , Reddehase M J , Koszinowski U H
Published: January 2020

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.
Evolution of influenza A virus nucleoprotein genes: implications for the origins of H1N1 human and classical swine viruses
Authors: Gorman O T , Bean W J , Kawaoka Y , Donatelli I , Guo Y J , Webster R G
Published: January 2020

A phylogenetic analysis of 52 published and 37 new nucleoprotein (NP) gene sequences addressed the evolution and origin of human and swine influenza A viruses. H1N1 human and classical swine viruses (i.e., those related to Swine/Iowa/15/30) share a single common ancestor, which was estimated to have occurred in 1912 to 1913. From this common ancestor, human and classical swine virus NP genes have evolved at similar rates that are higher than in avian virus NP genes (3.31 to 3.41 versus 1.90 nucleotide changes per year). At the protein level, human virus NPs have evolved twice as fast as classical swine virus NPs (0.66 versus 0.34 amino acid change per year). Despite evidence of frequent interspecies transmission of human and classical swine viruses, our analysis indicates that these viruses have evolved independently since well before the first isolates in the early 1930s. Although our analysis cannot reveal the original host, the ancestor virus was avianlike, showing only five amino acid differences from the root of the avian virus NP lineage. The common pattern of relationship and origin for the NP and other genes of H1N1 human and classical swine viruses suggests that the common ancestor was an avian virus and not a reassortant derived from previous human or swine influenza A viruses. The new avianlike H1N1 swine viruses in Europe may provide a model for the evolution of newly introduced avian viruses into the swine host reservoir. The NPs of these viruses are evolving more rapidly than those of human or classical swine viruses (4.50 nucleotide changes and 0.74 amino acid change per year), and when these rates are applied to pre-1930s human and classical swine virus NPs, the predicted date of a common ancestor is 1918 rather than 1912 to 1913. Thus, our NP phylogeny is consistent with historical records and the proposal that a short time before 1918, a new H1N1 avianlike virus entered human or swine hosts (O. T. Gorman, R. O. Donis, Y. Kawaoka, and R. G. Webster, J. Virol. 64:4893-4902, 1990). This virus provided the ancestors of all known human influenza A virus genes, except for HA, NA, and PB1, which have since been reassorted from avian viruses. We propose that during 1918 a virulent strain of this new avianlike virus caused a severe human influenza pandemic and that the pandemic virus was introduced into North American swine populations, constituting the origin of classical swine virus.
Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src
Authors: Wills J W , Craven R C , Weldon R A , Nelle T D , Erdie C R
Published: January 2020

The molecular mechanism by which retroviral Gag proteins are directed to the plasma membrane for the formation of particles (budding) is unknown, but it is widely believed that the MA domain, located at the amino terminus, plays a critical role. Consistent with this idea, we found that small deletions in this segment of the Rous sarcoma virus Gag protein completely blocked particle formation. The mutant proteins appear to have suffered only localized structural damage since they could be rescued (i.e., packaged into particles) when coexpressed with Gag proteins that are competent for particle formation. To our surprise, the effects of the MA deletions could be completely suppressed by fusing as few as seven residues of the myristylated amino terminus of the oncoprotein p60src to the beginning of the mutant Gag proteins. Particles produced by the chimeras were of the same density as the wild type. Two myristylated peptides having sequences distinct from that of p60src were entirely unable to suppress MA deletions, indicating that myristate alone is not a sufficient membrane targeting signal. We hypothesize that the amino terminus of p60src suppresses the effects of MA deletions by diverting the Rous sarcoma virus Gag protein from its normal site of assembly to the Src receptor for particle formation.
Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription
Authors: Cobrinik D , Aiyar A , Ge Z , Katzman M , Huang H , Leis J
Published: January 2020

A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure-compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 terminal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA.
Mutational analysis of the cellular receptor for poliovirus
Authors: Freistadt M S , Racaniello V R
Published: January 2020

To identify sequences of the cellular poliovirus receptor (PVR) involved in viral infection, mutant PVR cDNAs were constructed and assayed for biological activity in mouse L cells. To confirm that mutant PVRs reached the cell surface, an immunological tag, consisting of part of CH3 from human immunoglobulin G1, was engineered into the PVR. Deletion of PVR amino acids 256 to 320 or 385 to the carboxy terminus yielded receptors that were able to support poliovirus infection. PVRs lacking amino acids 40 to 136 or 137 to 256 were expressed at the cell surface but were not active as receptors for poliovirus. The results show that immunoglobulin-type domain 3 and the extreme carboxy terminus of the PVR are not required for viral receptor function, but sequences within the two amino-terminal domains contribute to the initiation of poliovirus infection.
The endogenous Mtv-8 provirus resides within the V kappa locus
Authors: Yang J N , Dudley J P
Published: January 2020

The endogenous Mtv-8 provirus previously has been mapped within approximately 0.52 centimorgan from several V kappa markers on mouse chromosome 6. Using Southern blotting and DNA from a recombinant backcross mouse from the C57BL/6 (Mtv-8 positive) and C58 (Mtv-8 negative) strains, Mtv-8 was localized to the same side of the crossover point as immunoglobulin kappa (Ig kappa)-V24 but on the opposite side of the crossover from Ig kappa-V10 and Ig kappa-V21. Molecular cloning and characterization of cellular DNA adjacent to Mtv-8 revealed a functional V kappa 9 gene approximately 4.6 kb downstream and in the same transcriptional orientation as the provirus. These data suggest that Mtv-8 is within the centromere-proximal portion of the V kappa locus.
Differential formation of disulfide linkages in the core antigen of extracellular and intracellular hepatitis B virus core particles
Authors: Jeng K S , Hu C P , Chang C M
Published: January 2020

Our understanding of the assembly of hepatitis B virus is still very limited. We present evidence to demonstrate that the HBc antigen formed oligomers through disulfide linkages in the extracellular hepatitis B virus core (HBc) particles. However, the intracellular HBV core particles did not contain disulfide-linked HBc antigens. Furthermore, the extracellular particles which had disulfide bonds were more stable than intracellular particles at pH 7.5 and 10 and in 3 M NaCl and 4 M urea. These data suggest that the formation of disulfide bonds in the HBc antigen is important for the integrity of the viral core particles.
Monoclonal antibodies to gp100 inhibit penetration of human herpesvirus 6 and polykaryocyte formation in susceptible cells
Authors: Foà-Tomasi L , Boscaro A , di Gaeta S , Campadelli-Fiume G
Published: January 2020

We report the derivation and properties of a monoclonal antibody (MAb 2E4) which neutralizes human herpesvirus 6 (HHV-6). MAb 2E4 precipitated from lysates of infected cells a glycosylated polypeptide 100,000 in apparent molecular weight and minor components of 80,000, and 32,500. The predominant reactive protein after a pulse was the 100,000-molecular-weight peptide designated as gp100. The smaller polypeptides appeared in the precipitate predominantly after a chase. MAb 2E4 neutralized HHV-6 infectivity in the presence and in the absence of complement, and it inhibited the penetration of virus into the cells. Addition of MAb 2E4 as late as 6 h postinfection inhibited the formation of large polykaryocytes typical of HHV-6-infected cells.
Induction of cellular transcription factors in trigeminal ganglia of mice by corneal scarification, herpes simplex virus type 1 infection, and explantation of trigeminal ganglia
Authors: Valyi-Nagy T , Deshmane S , Dillner A , Fraser N W
Published: January 2020

In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation.
Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses
Authors: Jin H , Elliott R M
Published: January 2020

A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.
Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge
Authors: Taylor J , Pincus S , Tartaglia J , Richardson C , Alkhatib G , Briedis D , Appel M , Norton E , Paoletti E
Published: January 2020

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.
Rotavirus spike structure and polypeptide composition
Authors: Anthony I D , Bullivant S , Dayal S , Bellamy A R , Berriman J A
Published: January 2020

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.
The 5' noncoding sequences from a less virulent Theiler's virus dramatically attenuate GDVII neurovirulence
Authors: Lipton H L , Calenoff M , Bandyopadhyay P , Miller S D , Dal Canto M C , Gerety S , Jensen K
Published: January 2020

RNA transcripts derived from recombinant chimeras between the highly virulent GDVII virus and the less virulent BeAn virus were constructed to study the molecular pathogenesis of Theiler's murine encephalomyelitis virus infection. The presence of the BeAn 5' noncoding sequences in chimera 2 (BeAn 5' noncoding sequences joined with the GDVII nucleotides encoding the polyprotein and present in the 3' end) resulted in dramatic attenuation of GDVII neurovirulence and development of poliomyelitis in mice. This reduced neurovirulence was associated with slower virus growth and lower peak titers in the brain and spinal cord than with parental GDVII virus replication. On the other hand, the sites of replication following chimera 2 infection were the same as those seen in GDVII-infected mice; the distribution of virus antigen and histopathological changes indicated that chimera 2 replicates in neurons in the brain, e.g., in the neocortex, hippocampus, caudate putamen, and brain stem, as well as in anterior-horn cells in the spinal cord. Chimera 2 was efficiently cleared from the mouse central nervous system by day 30 postinfection, in marked contrast to the persistence of the BeAn parent in the central nervous system. This suggests that elements in the BeAn sequences that encode the polyprotein or are present in the 3' noncoding region are necessary for viral persistence. It is of interest that chimera 2-infected mice developed localized inflammatory, demyelinating lesions which were detected at day 28 postinfection but these lesions did not become larger with time. Thus, virus persistence appears to be required for maintenance and progression of immune-mediated demyelination. If the demyelinating lesions become sufficiently large, clinical signs and disease may develop.
Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein
Authors: Dillon P J , Lenz J , Rosen C A
Published: January 2020

A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.