bluuebunny/crossref_metadata_2025 huggingface dataset:
Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B
Authors: Gage P J , Levine M , Glorioso J C
Published: January 2020

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.
Analysis of nonnucleoside drug-resistant variants of human immunodeficiency virus type 1 reverse transcriptase
Authors: Boyer P L , Currens M J , McMahon J B , Boyd M R , Hughes S H
Published: January 2020

A number of chemically distinct nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been reported. Several lines of evidence, including the isolation of RT mutants that show cross resistance, suggest that, despite their structural diversity, many of these inhibitors bind to a common site on HIV-1 RT. We have recently reported that, on the basis of analyses of HIV-1/HIV-2 chimeras, the natural product calanolide A may interact with a different site or sites in HIV-1 RT. We have used BspMI cassette mutagenesis to prepare a collection of HIV-1 RT mutants that show resistance to the known members of the general class of nonnucleoside inhibitors. This collection of mutants can be used to determine whether a new drug will show cross resistance with known inhibitors and to define amino acid positions critical for the action of the drugs. The mutants were used to analyze calanolide A, 1H,3H-thiazolo[3,4-a]benzimidazole(4i), and the acyclic nucleoside analog 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine. These analyses suggest that all three drugs interact with HIV-1 RT within the previously defined common binding site for nonnucleoside inhibitors. However, the drugs respond differently to the panel of drug-resistant HIV-1 RTs, indicating that while the binding sites of the drugs overlap they are not identical.
Localization of the c-ets-2 transactivation domain
Authors: Chumakov A M , Chen D L , Chumakova E A , Koeffler H P
Published: January 2020

The human ets-2 proto-oncogene is one of the homologs of the v-ets gene, found in avian acutely transforming retrovirus E26 (D. Leprince, A. Gegonne, J. Call, C. de Taisne, A. Schneeberger, C. Lagrou, and D. Stehelin, Nature [London] 306:395-397, 1983; M. F. Nunn, P. H. Seeburg, C. Moscovici, and P. H. Duesberg, Nature [London] 306:391-395, 1983), which causes leukemia in chickens. We used the DNA-binding domain of yeast transcriptional activator GAL4 to locate the transactivation region of human ets-2. The transactivation domain of ets-2 was found in the N-terminal part of the protein, which is homologous to ets-1, and can be disrupted by deletion of a stretch of acidic amino acid residues. A transactivation-deficient mutant of ets-2 failed to transform Rat-1 cells and suppressed the transforming activity of coexpressed wild-type ets-2. A mutation in the putative DNA-binding region of ets-2 abolished transforming activity. We show that the motif crucial for ets-2 transactivation capability is necessary for transforming activity in Rat-1 cells. Mutant ets-2 protein that lacks the transactivation domain has a dominant negative effect on transformation by wild-type ets-2. We were unable to detect ets-2-dependent transcriptional regulation of several enhancers containing ets-binding motifs.
Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation
Authors: Davies R , Hicks R , Crook T , Morris J , Vousden K
Published: January 2020

The transforming function of human papillomavirus type 16 (HPV16) E7 has been shown to depend on activities additional to the ability to bind RB. In this paper we describe two further properties of E7 which may also contribute to transformation, an association with a histone H1 kinase at the G2/M phase of the cell cycle and an ability to bind the RB-related protein p107. The region of E7 identified previously as important for RB binding was found to be involved in the association with the kinase and complex formation with p107, although analysis of E7 point mutants within this region revealed a difference in the precise sequence requirement for RB and p107 binding. Association with the kinase activity correlated with the ability to bind RB, but the restriction of the kinase association to the G2/M phase of the cell cycle implies that this activity might not be directly mediated by RB binding. Since kinase-binding-deficient E7 mutants are also transformation defective, this may represent an independent function of E7 which plays a role in the G2/M phase of the cell cycle.
Biochemical, genetic, and functional analyses of the phosphorylation sites on the Epstein-Barr virus-encoded oncogenic latent membrane protein LMP-1
Authors: Moorthy R K , Thorley-Lawson D A
Published: January 2020

LMP-1 is the only Epstein-Barr virus-encoded latent protein known to have the properties of a transforming oncogene in rodent fibroblasts and the only latent protein, besides EBNA-1, detected in nasopharyngeal carcinoma and Hodgkin's lymphoma biopsies. LMP-1 is characterized by serine/threonine phosphorylation and rapid turnover (half-life, 2 to 3 h) due to specific proteolytic cleavage, which causes release of a phosphorylated C-terminal fragment (p25) into the cytoplasm. We used biochemical, functional, and mutational analyses to identify sites of phosphorylation. All of the phosphorylation sites detected lie in the C-terminal domain. In particular, we identified S-313 and T-324 as functionally important sites. Prevention of phosphorylation at S-313, by altering it to a glycine, prevented detectable phosphorylation of both LMP-1 and p25, indicating that it is a major site on both forms of the molecule. However, lack of detectable phosphorylation had no effect on p25 cleavage or on the ability of LMP-1 to transform Rat-1 fibroblasts. Alteration of S-313 to an aspartate resulted in a form of LMP-1 that was toxic to Rat-1 cells. Alteration of T-324 to a glycine residue had no detectable effect on the ability of LMP-1 to become serine phosphorylated or transform Rat-1 cells. Alteration of T-324 to a glutamate, however, inhibited all detectable phosphorylation and resulted in a form of LMP-1 that was unable to transform Rat-1 fibroblasts. These results are discussed in the context of a model in which LMP-1 function is modulated by phosphorylation and dephosphorylation at S-313 and T-324.
Relating structure to function in the hepatitis delta virus antigen
Authors: Lazinski D W , Taylor J M
Published: January 2020

Hepatitis delta virus expresses two forms of a single protein, the small (delta Ag-S) and large (delta Ag-L) antigens, which are identical except for an additional 19 residues present at the C terminus of delta Ag-L. While delta Ag-S is required to promote genome replication, delta Ag-L potently inhibits this process and also facilitates packaging of the viral genome by envelope proteins of the helper virus (hepatitis B virus). Regions within the antigens responsible for nuclear localization, RNA binding, and dimerization have been identified, yet it is not clear how these particular activities contribute to the ultimate replication and packaging phenotypes. Here we report the following findings. (i) Although the removal of the nuclear localization signal from either antigen resulted in significant cytoplasmic accumulation, both proteins still had access to the nucleus. As a consequence, no functional defect was observed with either mutant. (ii) The RNA-binding domain, although necessary for delta Ag-S function, could be deleted from delta Ag-L without compromising its ability to either inhibit replication or promote packaging. (iii) In contrast, the coiled-coil dimerization domain was required for both the activation of replication by delta Ag-S and the inhibition of replication by delta Ag-L. This region, with an additional 20 amino acids C-terminal to it, was necessary and sufficient to potently inhibit replication by interacting with the small antigen. (iv) The packaging property of delta Ag-L required a C-terminal Pro/Gly-rich region which is hypothesized to interact with the hepatitis B virus envelope proteins during the assembly process.
Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles
Authors: Rizvi T A , Panganiban A T
Published: January 2020

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.
Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity
Authors: Zingler K , Littman D R
Published: January 2020

Growth of macaque simian immunodeficiency virus (SIVmac) in certain cloned human T-cell lines, such as HUT.78, selects for isolates containing a premature stop codon within the cytoplasmic domain of the transmembrane envelope glycoprotein. In contrast, propagation of virus in macaques or in their cultured T cells favors replication of virus containing the full-length envelope glycoprotein. To elucidate the causes of this phenomenon, we used a human immunodeficiency virus pseudotyping system to assess the effects on infectivity of the cytoplasmic domains of envelope glycoproteins obtained from SIVmac1A11 and SIVmac239. These envelopes contain truncated and full-length cytoplasmic domains, respectively. By analyzing human immunodeficiency virus particles containing selectable genes pseudotyped with each glycoprotein or with chimeric derivatives, we found that truncation of the cytoplasmic domain resulted in a significant advantage in viral entry into HUT.78 T cells and CD4+ U87.MG glial cells. Truncation of the cytoplasmic domain significantly enhanced both envelope density on particles and envelope-mediated cell-to-cell fusion. It is likely that one or both of these effects contribute to the observed differences in infectivity and to the selection of virions with short cytoplasmic tails in human T cells.
Conservation of determinants for class II-restricted T cells within site E of influenza virus hemagglutinin and factors influencing their expression
Authors: Brown L E , White D O , Jackson D C
Published: January 2020

The determinants recognized by helper T cells specific for the site E region of H3 subtype influenza virus hemagglutinin (HA) have been defined by examining the reactivity of T-cell clones with sets of overlapping peptides of various lengths covering the site. Two overlapping sequences, TLIDALLG and LIDALLGDP, were identified as the minimal determinants for four of five representative clones. These sequences are located within a loop of the molecule closed by a disulfide bond and presumably require cleavage of this bond for interaction with the class II major histocompatibility molecule. In contrast, the determinant recognized by the fifth clone was dependent on the presence of an intact disulfide bond for its expression and could not be represented by a synthetic peptide homolog of the linear sequence. Both TLIDALLG and LIDALLGDP are conserved within all field strains of the H3 subtype. Nevertheless, recognition of these sequences by the T-cell clones is affected by the glycosylation pattern of the hemagglutinin and by residues lying outside the minimal determinant. Three distinct clones directed towards the sequence LIDALLGDP were remarkably similar in their pattern of response to a set of synthetic analogs of the determinant, suggesting that residues of the T-cell receptor other than those contacting the minimal determinant may be responsible for the different specificities observed for these clones with different field strains of virus.
Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression
Authors: Kashanchi F , Duvall J F , Lindholm P F , Radonovich M F , Brady J N
Published: January 2020

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription probably play an important role in initiation and maintenance of virus replication. We have identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]) at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. The basal promoter strength of constructs that contained deletions in the R/U5 region of the HTLV-I long terminal repeat were analyzed by chloramphenicol acetyltransferase assays following transfection of Jurkat T cells. We consistently observed a 10-fold decrease in basal promoter activity when sequences between +202 to +246 were deleted. By reverse transcriptase polymerase chain reaction RNA analysis, we confirmed that the drop in chloramphenicol acetyltransferase activity was paralleled by a decrease in the level of steady-state RNA. DRE 1 did not affect the level of Tax1 transactivation. Using a gel shift assay, we have purified a highly enriched fraction that could specifically bind DRE 1. This DNA affinity column fraction contained four detectable proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: p37, p50, p60, and p100. The affinity column fraction stimulated HTLV-I transcription approximately 12-fold in vitro. No effect was observed with the human immunodeficiency virus or adenovirus major late promoters. Following renaturation of the proteins isolated from an SDS-containing gel, p37, but not the other protein fractions, was able to specifically bind to DRE 1.
Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen
Authors: Kimman T G , Bianchi A T , Wensvoort G , de Bruin T G , Meliefste C
Published: January 2020

T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect. Therefore, little is known about cellular immunity to HCV. In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs. We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals. However, such cells proliferated in vitro upon stimulation with viable HCV antigen. The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV. Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response. The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain. A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains. This immunological phenomenon may mask a specific T-cell response to the virus.
Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase
Authors: Colman P M , Hoyne P A , Lawrence M C
Published: January 2020

A model is proposed for the three-dimensional structure of the paramyxovirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the enzyme-active center of influenza virus neuraminidase. The influenza virus enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.
Evidence for a functional subclass of the RTVL-H family of human endogenous retrovirus-like sequences
Authors: Wilkinson D A , Goodchild N L , Saxton T M , Wood S , Mager D L
Published: January 2020

We report the discovery of a subgroup of RTVL-H human endogenous retroviral elements, designated RTVL-Hp, that is intact in the pol region which is deleted in the vast majority of RTVL-H elements. As a consequence, RTVL-Hp elements contain critical functional domains in their pol region that other RTVL-H elements lack. We estimate that the haploid genomes of humans, apes, and Old World monkeys contain 50 to 100 copies of RTVL-Hp elements and 800 to 1,000 deleted sequences. The major amplification of deleted elements appears to have occurred after the divergence of Old World and New World monkeys, since we have obtained evidence that a few intact RTVL-Hp elements, but no deleted forms, are present in marmoset DNA. Using the polymerase chain reaction coupled with a direct screen for open reading frames, we have isolated fragments from four RTVL-Hp elements amplified from human DNA that contain an open reading frame throughout a region of pol that is disrupted by diagnostic mutations in all other RTVL-H sequences that we had previously analyzed. Northern (RNA) hybridization analysis shows that unit-length RTVL-Hp transcripts are expressed in the human teratocarcinoma cell line Tera-1. Together, the results presented here suggest that a small functional subfamily of RTVL-H elements is present in the human genome.
RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol
Authors: Cho M W , Richards O C , Dmitrieva T M , Agol V , Ehrenfeld E
Published: January 2020

The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3Dpol, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the gamma-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3Dpol is not a property shared by all polymerases.
Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens
Authors: Calvert J G , Nazerian K , Witter R L , Yanagida N
Published: January 2020

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.
A small highly basic protein is encoded in overlapping frame within the P gene of vesicular stomatitis virus
Authors: Spiropoulou C F , Nichol S T
Published: January 2020

Vesicular stomatitis virus (VSV) has served for several decades as the prototype rhabdovirus and a model RNA virus. Extensive studies upheld the original view of VSV genetics with simply five genes (N, P, M, G, and L), each encoding a single unique protein. We now report the first unambiguous demonstration of the existence of an additional unique protein encoded in an overlapping frame within the virus P gene. Experiments using antipeptide sera specific for the predicted second open reading frame have demonstrated the synthesis of two N-terminally nested forms of the protein in virus-infected cells. The major form is 55 amino acids in length, whereas the minor form has 10 additional N-terminal amino acids. Ribosome initiation of synthesis of these proteins appears to occur at AUG codons, 68 and 41 bases, respectively, downstream of the P protein AUG initiation codon. The proteins are found in the cytoplasm of the infected cell but are undetectable in purified virions, consistent with their being nonstructural proteins. Both the major and minor forms of the protein are highly basic and arginine rich, reminiscent of the C and C' proteins encoded in overlapping frame close to the 5' terminus of the P mRNA of several paramyxoviruses. The potential to encode small, highly basic proteins within the P mRNA 5' terminus is highly conserved among the vesiculoviruses.
Transcripts from the Epstein-Barr virus BamHI A fragment are detectable in all three forms of virus latency
Authors: Brooks L A , Lear A L , Young L S , Rickinson A B
Published: January 2020

An unexpected feature of the latency II form of Epstein-Barr virus (EBV) infection seen in the epithelial tumor nasopharyngeal carcinoma (NPC) is the presence of spliced polyadenylated RNAs encoded from the BamHI A fragment of the viral genome and running in the opposite orientation to several BamHI-A lytic cycle genes. The importance of these BamHI-A transcripts and the specificity of their association with NPC remain to be determined. In this study, we examined the extent to which such RNAs are present in other transcriptionally distinct forms of EBV latency seen in B cells. Two independent assays of BamHI-A transcription were employed: amplification across defined splice junctions in cDNAs, using the polymerase chain reaction, and in situ hybridization with a radiolabeled riboprobe specific for a putative open reading frame downstream of these splice junctions. Such methods, which easily detected BamHI-A RNAs in fresh NPC biopsies and transplantable NPC lines, also revealed consistent expression of these transcripts in all EBV-positive Burkitt's lymphoma cell lines displaying the highly restricted latency I form of infection (BamHI-F promoter usage) as well as in all EBV-transformed lymphoblastoid cell lines (LCLs) displaying the latency III form of infection (BamHI-C/W promoter usage). Expression in established LCLs, occurring irrespective of virus producer status, was not a consequence of continued in vitro passage; thus, appropriately spliced BamHI-A transcripts could be amplified from normal B cells within 1 day of their experimental infection in vitro, along with BamHI-C/W promoter-initiated but not BamHI-F promoter-initiated mRNAs. In situ hybridization both on Burkitt's lymphoma cell lines and on LCLs showed that essentially every cell contained BamHI-A transcripts, although at levels apparently lower than those observed in NPC. We conclude that expression of the BamHI-A RNAs is a consistent feature shared by all known forms of latent EBV infection.
Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection
Authors: Zhang L Q , MacKenzie P , Cleland A , Holmes E C , Brown A J , Simmonds P
Published: January 2020

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.
Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro
Authors: Fredman J N , Engler J A
Published: January 2020

The adenovirus precursor to the terminal protein (pTP), expressed in a vaccinia virus expression system or in native adenovirus, was assayed for its ability to interact with the nuclear matrix. Biochemical function was measured by determining the relative amount of pTP protein or of adenovirus DNA that remained associated with the nuclear matrix after extensive washing. pTP was retained on the matrix whereas beta-galactosidase was not, as assayed by quantitative immunoblot analysis. Nuclear matrix isolated from adenovirus-infected HeLa cells retained bound adenovirus DNA even when washed with 1 M guanidine hydrochloride; this interaction could be inhibited by added purified pTP protein. Analogous experiments with matrix isolated from HeLa cells infected with a recombinant vaccinia virus that expressed pTP showed a similar retention of pTP protein; this association could also be inhibited by added pTP protein. Binding of pTP to nuclear matrix isolated from uninfected cells was saturable, with an apparent Kd of 250 nM and an estimated 2.8 x 10(6) sites for pTP binding per cell nucleus. The association of pTP with matrix is postulated to help direct adenovirus replication complexes to the appropriate locale within the nucleus.
The vaccinia virus 14-kilodalton fusion protein forms a stable complex with the processed protein encoded by the vaccinia virus A17L gene
Authors: Rodriguez D , Rodriguez J R , Esteban M
Published: January 2020

The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the VV A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several VV structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INV.
Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses
Authors: Monroe S S , Jiang B , Stine S E , Koopmans M , Glass R I
Published: January 2020

We report the sequence of the subgenomic RNA of human astrovirus serotype 2. This 2,484-nucleotide RNA contains a single open reading frame, which encodes a protein with a predicted molecular mass of 88 kDa. We propose that this protein is the 90-kDa capsid precursor observed in infected cells. The deduced protein sequence does not contain conserved amino acid patterns reported for the capsid proteins of picornaviruses or caliciviruses, consistent with the classification of astroviruses as a new family of RNA viruses, designated Astroviridae.
An immortalized human fibroblast cell line is permissive for human cytomegalovirus infection
Authors: Compton T
Published: January 2020

Human foreskin fibroblasts (HFF) were immortalized via retrovirus-mediated gene transfer of the E6 and E7 genes of human papillomavirus type 16. An immortalized fibroblast (IF) cell line which was morphologically akin to the parental cell line was isolated. The IF cell line was evaluated for permissiveness to human cytomegalovirus (HCMV) infection after the IF cell line surpassed the normal passage limitation of diploid fibroblasts. Western immunoblot analysis of representative HCMV-encoded immediate-early (72-kDa), early (gB), and late (gH) gene products demonstrated that the IF cell line produced these proteins analogous to those produced by the parental HFF cells. Similar quantities of infectious virus were produced in the IF and HFF cell lines as determined in one-step growth curve experiments. Compared with the HFF cells, morphologically identical plaques were produced in the IF cell line in approximately 10 to 12 days postinfection. These findings indicate that fibroblast cell lines immortalized with transforming genes of human papillomavirus retain complete permissiveness to HCMV infection and support plaque formation. The IF cell line will be useful for future genetic analysis of HCMV.
The herpes simplex virus type 1 reactivation function lies outside the latency-associated transcript open reading frame ORF-2
Authors: Farrell M J , Hill J M , Margolis T P , Stevens J G , Wagner E K , Feldman L T
Published: January 2020

The latency-associated transcription unit has been shown to be important for in vivo reactivation of herpes simplex virus from the latent state. A recombinant virus was constructed to alter the largest open reading frame in this region. This virus had a wild-type reactivation phenotype, suggesting that herpes simplex virus does not require a protein function from this reading frame for efficient reactivation from latency.
Effect of amino acid changes in the V1/V2 region of the human immunodeficiency virus type 1 gp120 glycoprotein on subunit association, syncytium formation, and recognition by a neutralizing antibody
Authors: Sullivan N , Thali M , Furman C , Ho D D , Sodroski J
Published: January 2020

The contributions of the first and second variable regions of the human immunodeficiency virus type 1 gp120 glycoprotein to envelope glycoprotein structure, function, and recognition by a neutralizing antibody were studied. Several mutants with substitutions in the V2 loop demonstrated complete dissociation of the gp120 and gp41 glycoproteins, suggesting that inappropriate changes in V2 conformation can affect subunit assembly. Some glycoproteins with changes in V1 or V2 were efficiently expressed on the cell surface and were able to bind CD4 but were deficient in syncytium formation and/or virus entry. Recognition of gp120 by the neutralizing monoclonal antibody G3-4 was affected by particular substitutions affecting residues 176 to 184 in the V2 loop. These results suggest that the V1/V2 variable regions of the human immunodeficiency virus type 1 gp120 glycoprotein play a role in postreceptor binding events in the membrane fusion process and can act as a target for neutralizing antibodies.
Identification of a new transcriptional unit that yields a gene product within the unique sequences of the short component of the herpes simplex virus 1 genome
Authors: Georgopoulou U , Michaelidou A , Roizman B , Mavromara-Nazos P
Published: January 2020

The herpes simplex virus genome 1 consists of two unique stretches, long (UL) and short (U(S)), each flanked by inverted repeat sequences. The U(S) sequence has been previously reported to contain 12 open reading frames designated U(S)1 through U(S)12. This report demonstrates the existence of a 13th open reading frame within the U(S) sequence, designated U(S)8.5. The U(S)8.5 sequence is located between, and overlaps in part with, the domains of the U(S)8 and U(S)9 genes. Its transcription is initiated within the coding sequence of U(S)8, and its transcript decays earlier than that of U(S)8. On the basis of the size of its RNA (1.2 kb) and map position, it is likely that the U(S)8.5 transcript is 3' coterminal with the U(S)8 and U(S)9 mRNAs at the single polyadenylation signal which serves these genes. The nucleotide sequence of the U(S)8.5 open reading frame predicts that its product is a 151-amino-acid basic, hydrophilic polypeptide. To determine whether the U(S)8.5 encodes a protein, a sequence encoding 23 amino acids that contains an epitope reacting with a known monoclonal antibody to human cytomegalovirus protein was inserted in frame after the predicted fifth codon of the U(S)8.5 gene. The recombinant virus carrying this epitope induced the synthesis of a protein reactive with the monoclonal antibody in immunoblots. The tagged protein localized in nucleoli of cells infected with the recombinant virus.
The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals
Authors: Ryon J J , Fixman E D , Houchens C , Zong J , Lieberman P M , Chang Y N , Hayward G S , Hayward S D
Published: January 2020

Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid.
Characterization of the major locus of immediate-early genes of rat cytomegalovirus
Authors: Sandford G R , Ho K , Burns W H
Published: January 2020

A major locus of rat cytomegalovirus (RCMV) immediate-early (IE) RNA transcription was identified. A cDNA library from rat embryo fibroblasts infected with RCMV under IE conditions was constructed and screened by using appropriate RCMV DNA probes, revealing at least two IE genes (IE1 and IE2) transcribed from this locus by differential splicing. The first three exons (the first is noncoding) are spliced to exon 4 to form IE1 and to exon 5 to form IE2. The structural organization of the RCMV major IE region is therefore similar to that of human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). When we compared the predicted amino acid sequences of the IE1 proteins of RCMV, HCMV, and MCMV, no areas of homology were found across all three proteins, while a few small areas of homology were found between RCMV IE1 and MCMV IE1. In contrast, large areas of homology were found across the carboxyl half of RCMV IE2, HCMV IE2, and MCMV ie3 proteins. In addition, similarities were found at the beginning of exon 5 of RCMV and MCMV. The possible significance of these conserved regions is discussed. Dinucleotide frequency analysis demonstrated a decrease in CpG frequency over the IE region. The IE gene products were able to transactivate heterologous promoters.
Cottontail rabbit papillomavirus L1 protein-based vaccines: protection is achieved only with a full-length, nondenatured product
Authors: Lin Y L , Borenstein L A , Ahmed R , Wettstein F O
Published: January 2020

Papillomas induced by the cottontail rabbit papillomavirus (CRPV) progress at a high frequency to carcinomas. In this regard, CRPV and its tumors can serve as an animal model for highly oncogenic human papillomaviruses. We have previously shown that immunization with major structural protein L1 elicits neutralizing antibodies and protects rabbits from papilloma development (Y.-L. Lin, L.A. Borenstein, R. Selvakumar, R. Ahmed, and F.O. Wettstein, Virology 187:612-619, 1992). In this study, we demonstrated that vaccination with the TrpE-L1 fusion protein not only protected rabbits from papilloma development but also prevented latent infection. This was indicated by the failure to amplify CRPV sequences by polymerase chain reaction in biopsies from infection sites of immunized animals. Furthermore, we showed that TrpE-L1 immunization protected rabbits from papilloma formation induced by virus but not from that induced by viral DNA. To explore the possibility of developing vaccines based on L1 subfragments, we mapped the linear L1 epitopes recognized by TrpE-L1-immunized rabbits and by virus-infected rabbits resistant to superinfection. Sera from papilloma-bearing rabbits reacted with one major epitope located at the carboxy-terminal end of L1, between amino acids (aa) 480 and 505. A second epitope, and in some animals a third one, was located in the amino-terminal region, between aa 78 and 101, as well as between aa 37 and 62. Sera from TrpE-L1-immunized animals recognized only one major epitope, located between aa 6 and 37. Immunization of rabbits with L1 subfragment fusion proteins led to seroconversion, but no neutralizing antibodies were produced and the animals were not protected against papilloma formation. The data indicate that a successful papillomavirus vaccine must be based on immunization with full-length native L1 and that further simplification to smaller peptides containing major linear epitopes is not feasible.
Transgenic Fv-4 mice resistant to Friend virus
Authors: Limjoco T I , Dickie P , Ikeda H , Silver J
Published: January 2020

Fv-4 is a mouse gene that confers resistance to infection with ecotropic retroviruses. A candidate Fv-4 gene was cloned previously and found to resemble the 3' half of a murine leukemia virus (MuLV). To study the effect of this gene in vivo, we generated two transgenic mouse strains carrying the Fv-4 env gene under control of its presumed natural promoter, a cellular sequence unrelated to retroviruses. Transgenic progeny expressed a 3-kb Fv-4 env RNA in all of the organs and tissues examined, as well as an Fv-4 envelope antigen on the surface of thymocytes and spleen cells, similar to mice carrying the natural Fv-4 gene. One of the two transgenic strains (designated Fv4-2) expressed three to nine times as much transgene RNA and protein as the other strain (Fv4-11). When challenged with a Friend virus complex containing up to 10(4) XC PFU of Friend MuLV, Fv4-2 mice were completely resistant to development of splenomegaly and had no detectable ecotropic virus in the spleen or blood, confirming that the cloned Fv-4 gene is responsible for resistance to ecotropic MuLV in vivo. In contrast, Fv4-11 mice were only partially resistant, developing viremia and splenomegaly at the highest inoculum dose but recovering from viremia several weeks after inoculation with 10-fold less virus. The phenotype of recovery from viremia in Fv4-11 mice was unexpected and suggests that low levels of expression of the Fv-4 gene enhance the effectiveness of the immune response.
Repression of RNA polymerase III transcription by adenovirus E1A
Authors: Sollerbrant K , Akusjärvi G , Svensson C
Published: January 2020

Adenovirus E1A encodes two major proteins of 289 and 243 amino acids (289R and 243R), which both have transcription regulatory properties. E1A-289R is a transactivator whereas E1A-243R primarily functions as a repressor of transcription. Here we show that E1A repression is not restricted to RNA polymerase II genes but also includes the adenovirus virus-associated (VA) RNA genes. These genes are transcribed by RNA polymerase III and have previously been suggested to be the target of an E1A-289R-mediated transactivation. Surprisingly, we found that during transient transfection both E1A proteins repressed VA RNA transcription. E1A repression of VA RNA transcription required both conserved regions 1 and 2 and therefore differed from the E1A-mediated inhibition of simian virus 40 enhancer activity which primarily required conserved region 1. The repression was counteracted by the E1B-19K protein, which also, in the absence of E1A, enhanced the accumulation of VA RNA. Importantly, we show that efficient VA RNA transcription requires expression of both E1A and the E1B-19K protein during virus infection.
Hepatitis B virus (HBV) binding factor in human serum: candidate for a soluble form of hepatocyte HBV receptor
Authors: Budkowska A , Quan C , Groh F , Bedossa P , Dubreuil P , Bouvet J P , Pillot J
Published: January 2020

A hepatitis B virus (HBV) binding factor (HBV-BF) was identified in normal human serum interacting with the pre-S1 and pre-S2 epitopes of the viral envelope located within the protein domains involved in recognition of hepatocyte receptor(s). This molecule was characterized as a 50-kDa glycoprotein showing an isoelectric point of 7.13 with a biological activity depending on its native molecular conformation and on intact sulfhydryl bonds. Monoclonal antibodies to HBV-BF recognized a membrane component of the normal human liver whereas they were unreactive with hepatocyte membranes of other species and with those of the HepG2 cell line. These results suggest that the HBV-BF represents a soluble fragment of the membrane component and can be related to the HBV receptor mediating attachment of HBV to human liver cells.