bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic equilibrium between vesicular stomatitis virus glycoprotein monomers and trimers in the Golgi and at the cell surface
Authors: Zagouras P , Rose J K
Published: January 2020

Previous studies have shown that trimers of the vesicular stomatitis virus glycoprotein (VSV G protein) are in rapid equilibrium with monomeric subunits after folding and assembly in the endoplasmic reticulum (ER). To determine whether G protein trimers were in equilibrium with monomers in other cellular compartments, we studied heterotrimer formation between VSV G protein and a mutant G protein (G mu protein) containing a 3-amino-acid cytoplasmic domain replacing the normal 29-amino-acid domain. The G mu protein is transported from the ER much more slowly than G protein, although both G and G mu proteins form trimers rapidly in the ER. In coexpression experiments, we observed that VSV G protein molecules exited the ER about sixfold faster than G mu protein molecules, and we observed no heterotrimer formation in the ER, probably because of rapid reassortment of the mutant and wild-type trimers. However, heterotrimer formation between the two proteins was observed after long chase periods that allowed time for trimers of the mutant protein to reach the plasma membrane and reassort with the G protein subunits. Additional studies showed that heterotrimers of the two proteins could form in the Golgi or in the ER if exit of the G protein from either compartment was blocked.
Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription
Authors: Kleinberger T , Shenk T
Published: January 2020

Adenovirus E4orf4 protein was previously shown to counteract transactivation of junB by cyclic AMP (cAMP) and E1A protein. It was also shown to cause hypophosphorylation of E1A and c-Fos proteins. Here we show that the E4orf4 protein associates with protein phosphatase 2A. All three subunits of the phosphatase are present in the complex, and the B subunit interacts directly with the viral protein. The complex possesses a phosphatase activity typical of protein phosphatase 2A, and the phosphatase mediates the E4orf4-induced down regulation of junB transcription. Thus, adenovirus E4orf4 protein recruits protein phosphatase 2A into a signal transduction pathway initiated by cAMP and E1A protein.
Complete nucleotide sequence of a highly divergent human T-cell leukemia (lymphotropic) virus type I (HTLV-I) variant from melanesia: genetic and phylogenetic relationship to HTLV-I strains from other geographical regions
Authors: Gessain A , Boeri E , Yanagihara R , Gallo R C , Franchini G
Published: January 2020

The high prevalences of antibodies against human T-cell leukemia (lymphotropic) virus type I (HTLV-I) reported for remote populations in Papua New Guinea and the Solomon Islands and for some aboriginal populations in Australia have been verified by virus isolation. Limited genetic analysis of the transmembrane portion (gp21) of the envelope gene of these viruses indicates the existence of highly divergent HTLV-I strains in Melanesia. Here, we report the complete nucleotide sequence of an HTLV-I isolate (designated HTLV-IMEL5) from the Solomon Islands. The overall nucleotide divergence of HTLV-IMEL5 from the prototype HTLV-IATK was approximately 8.5%. The degree of variability in the amino acid sequences of structural genes ranged between 3 and 11% and was higher (8.5 to 25%) for the regulatory (tax and rex) genes and the other genes encoded by the pX region. Since HTLV-IMEL5 was as distantly related to HTLV-II as to the other known HTLV-I strains, it could not have arisen from a reocmbinational event involving HTLV-II but rather might be an example of independent viral evolution in this remote population. These data provide important insights and raise new questions about the origin and global dissemination of HTLV-I.
Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS
Authors: Talarico C L , Phelps W C , Biron K K
Published: January 2020

Patients with AIDS often experience recurrent infections with varicella-zoster virus (VZV) requiring repeated or prolonged treatment with acyclovir (ACV), which may lead to the development of ACV resistance. The ACV resistance of isolates recovered from such patients is associated with diminished VZV thymidine kinase (TK) function. We determined the nucleotide sequences of the TK genes of 12 ACV-resistant VZV strains purified from nine patients with AIDS. Five VZV strains contained nucleotide deletions in their TK genes, introducing a premature termination codon which is expected to result in the production of a truncated protein. No detectable full-length TK protein could be immunoprecipitated from extracts of cells infected with these virus strains. These TK-deficient strains were cross resistant to the TK-dependent antiviral agents ACV, 9-(4-hydroxy-3-hydroxymethylbutyl-yl)guanine (penciclovir), and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil (BVaraU). The remaining seven strains each contained a nucleotide change that resulted in an amino acid substitution in the TK protein. These substitutions occurred throughout the TK protein, namely, in the ATP-binding site, the nucleoside-binding site, between the two binding sites, and at the carboxy terminus of the protein. We determined the effects of these mutations on the stability of TK protein expression in virus-infected cells and on the sensitivity of mutants to the TK-dependent antiviral agents ACV, BVaraU, and penciclovir.
NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells
Authors: Naeger L K , Salomé N , Pintel D J
Published: January 2020

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.
v-erbA acts on retinoic acid receptors in immature avian erythroid cells
Authors: Sande S , Sharif M , Chen H , Privalsky M
Published: January 2020

The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes an aberrant version of a gene for a thyroid hormone receptor (c-erbA) and functions in neoplasia by blocking erythroid differentiation and altering the growth properties of fibroblasts. The v-erbA gene has been proposed to act as a dominant negative allele, functioning by interfering with the actions of its normal cell homologs, the thyroid hormone receptors. The v-erbA protein can also, however, interfere with the actions of other members of the nuclear hormone receptor family, and it has been proposed that interference with a retinoic acid-mediated response may be a crucial determinant of v-erbA's function in the cancer cell. Here we report that the ability of v-erbA to interfere with retinoic acid receptor (RAR) action extends to the neoplastic erythroid cell and that v-erbA can inhibit transcriptional activation by all three isoforms (alpha, beta, and gamma) of RAR. Overexpression of RAR-alpha was found to partially overcome the presumptive v-erbA block to transcription in the erythroleukemic cell. These results are consistent with our hypothesis that v-erbA can act in neoplasia by interfering with a retinoic acid-mediated signal transduction pathway.
Role of tumor necrosis factor alpha in activation and replication of the tat-defective human immunodeficiency virus type 1
Authors: Popik W , Pitha P M
Published: January 2020

Transcription of human immunodeficiency virus type 1 (HIV-1) depends on the function of the virus-encoded regulatory protein Tat, which interacts with the specific Tat response (TAR) element present in the leader sequence of all HIV-1 RNAs. In this study, we examined whether tumor necrosis factor alpha (TNF-alpha) can replace the requirement for a functional Tat protein. We found that TNF-alpha can induce expression of a latent, tat-defective virus and support its replication both in T cells and in primary mononuclear cells. Analysis of the transcriptional rate of the tat-defective HIV-1 transcriptional unit indicates that TNF-alpha stimulates the initiation of transcription but, in contrast to Tat protein, does not significantly reduce transcriptional polarity. Interestingly, we found that the processing of viral precursor proteins is altered in the absence of Tat. We propose that TNF-alpha-mediated induction of HIV-1 plays an essential role in the early stages of the virus life cycle and in viral latency.
TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins
Authors: Rhim H , Rice A P
Published: January 2020

Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein.
Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies
Authors: Ohlin M , Sundqvist V A , Mach M , Wahren B , Borrebaeck C A
Published: January 2020

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.
Papillomavirus E7 protein binding to the retinoblastoma protein is not required for viral induction of warts
Authors: Defeo-Jones D , Vuocolo G A , Haskell K M , Hanobik M G , Kiefer D M , McAvoy E M , Ivey-Hoyle M , Brandsma J L , Oliff A , Jones R E
Published: January 2020

Human papillomaviruses (HPVs) are the etiologic agents responsible for benign epithelial proliferative disorders including genital warts and are a contributory factor in the pathogenesis of cervical cancer. HPVs demonstrate strict species and cell-type specificity, which is manifested by the inability of these viruses to induce disease in any species other than humans. The natural history of HPV infection in humans is closely mimicked by cottontail rabbit papillomavirus (CRPV) infection in domestic laboratory rabbits. The CRPV E7 gene is known to play an essential role in virus-mediated induction of papillomas. We now show by mutational analysis that the CRPV E7 protein's biochemical and biological properties, including binding to the retinoblastoma suppressor protein (pRB), transcription factor E2F transactivation of the adenovirus E2 promoter, disruption of pRB-E2F complexes, and cellular transformation as measured by growth in soft agar, mimic those of the HPV E7 protein. Intradermal injection of CRPV DNA lacking E7 gene sequences critical for the binding of the CRPV E7 protein to pRB induced papillomas in rabbits. These studies indicate that E7 protein binding to pRB is not required in the molecular pathogenesis of virally induced warts and suggest that other properties intrinsic to the E7 protein are necessary for papilloma formation.
Biologic importance of neuraminidase stalk length in influenza A virus
Authors: Castrucci M R , Kawaoka Y
Published: January 2020

To investigate the biologic importance of the neuraminidase (NA) stalk of influenza A virus, we generated mutant viruses of A/WSN/33 (H1N1) with stalks of various lengths (0 to 52 amino acids), by using the recently developed reverse genetics system. These mutant viruses, including one that lacked the entire stalk, replicated in tissue culture to the level of the parent virus, whose NA stalk contains 24 amino acid residues. In eggs, however, the length of the stalk was correlated with the efficiency of virus replication: the longer the stalk, the better the replication. This finding indicates that the length of the NA stalk affects the host range of influenza A viruses. The NA stalkless mutant was highly attenuated in mice; none of the animals died even after intranasal inoculation of 10(6) PFU of the virus (the dose of the parent virus required to kill 50% of mice was 10(2.5) PFU). Moreover, the stalkless mutant replicated only in the respiratory organs, whereas the parent virus caused systemic infection in mice. Thus, attenuation of the virus with the deletion of the entire NA stalk raises the possibility of its use as live vaccines.
Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein-Barr virus R transactivator
Authors: van Santen V L
Published: January 2020

Immediate-early (IE) RNA 2, the less abundant of two bovine herpesvirus 4 (BHV-4) RNAs detected in Madin-Darby bovine kidney cells infected in the presence of cycloheximide, is a 1.8-kb cytoplasmic polyadenylated RNA transcribed from the 8.3-kb HindIII fragment F. The structure of IE RNA 2 has been determined by S1 nuclease and exonuclease VII mapping, primer extension analysis, and sequencing of a partial cDNA. IE RNA 2 consists of a short, approximately 60-nucleotide 5' exon spliced to a 1.8-kb 3' exon. DNA sequence analysis revealed an open reading frame encoding 551 amino acids with sequence homology to the Epstein-Barr virus (EBV) R transactivator and its homolog in herpesvirus saimiri, HVS.R.IE 2 and HVS.R show higher homology to each other than to the EBV R transactivator. The homology is highest in the approximately 320 amino-terminal amino acids. All three proteins have acidic carboxyl termini but have little amino acid sequence homology in this region. In transient expression cotransfection assays, IE 2 activated expression from the BHV-4 early promoter-regulatory region of the major DNA-binding protein homolog over 100-fold in bovine turbinate cells. IE 1 was not necessary for this transactivation and did not augment it. However, IE 2 did not transactivate EBV or herpesvirus saimiri early promoter-regulatory regions that are transactivated by the EBV R transactivator or HVS.R.
Correlation of specific virus-astrocyte interactions and cytopathic effects induced by ts1, a neurovirulent mutant of Moloney murine leukemia virus
Authors: Shikova E , Lin Y C , Saha K , Brooks B R , Wong P K
Published: January 2020

ts1 is a highly neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus TB (MoMuLV-TB). We previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-->Ile, in precursor envelope protein gPr80env. This Val-25-->Ile substitution apparently renders gPr80env inefficient for transport from the endoplasmic reticulum to the Golgi apparatus. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to the accumulation of gPr80env in the endoplasmic reticulum. Since endothelial and glial cells are targets of ts1 infection in the central nervous system, we established primary endothelial and astrocyte cultures to investigate the mechanism of cell killing caused by ts1. A continuous cell line, TB, was used as a control. Our results showed that both ts1 and MoMuLV-TB replicated and induced a cytopathic effect in astrocyte cultures, albeit to different degrees; ts1 appeared to be more lethal than MoMuLV-TB. On the other hand, ts1 and MoMuLV-TB infections of endothelial or TB cells were not cytopathic. The cytopathic effect in infected astrocytes correlated with the inefficiency of gPr80env transport and the intracellular accumulation of gPr80env as well as aberrant virus particles.
Functional analysis of the herpes simplex virus UL42 protein
Authors: Digard P , Chow C S , Pirrit L , Coen D M
Published: January 2020

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.
The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks
Authors: Chen H S , Kaneko S , Girones R , Anderson R W , Hornbuckle W E , Tennant B C , Cote P J , Gerin J L , Purcell R H , Miller R H
Published: January 2020

All mammalian hepadnaviruses possess a gene, termed X, that encodes a protein capable of transactivating virus gene expression. The X gene overlaps the polymerase and precore genes as well as two newly identified open reading frames (ORFs) termed ORF5 and ORF6. In this investigation, we examined whether ORF5, ORF6, and the X gene were important for the replication of woodchuck hepatitis virus (WHV) in susceptible woodchucks. First, we investigated whether proteins were produced from ORF5 and ORF6 by in vitro translation of appropriate viral transcripts, searched for antibodies against the putative proteins in the sera of animals infected with wild-type virus, and looked for an antisense WHV transcript, necessary for expression of a protein from ORF6, in the livers of acutely or chronically infected woodchucks. All such experiments yielded negative results. Next, we used oligonucleotide-directed mutagenesis to introduce termination codons into ORF5 and ORF6 at two locations within each ORF. Adult woodchucks in groups of three were transfected with one of the four mutant genomes. All of these woodchucks developed WHV infections that were indistinguishable from those of animals transfected with the wild-type WHV recombinant. Polymerase chain reaction amplification and direct DNA sequencing confirmed that reversion of the mutants to a wild-type genotype did not occur. Taken together, these data indicate that ORF5 and ORF6 are not essential for virus replication and are unlikely to represent authentic genes. Finally, we generated five WHV X-gene mutants that either removed the initiation codon for protein synthesis or truncated the carboxyl terminus of the protein by 3, 16, 31, or 52 amino acids. Groups of three adult woodchucks were transfected with one of the five X-gene mutants. Only the mutant that possessed an X gene lacking 3 amino acids from the carboxyl terminus was capable of replication within the 6-month time frame of the experiment. In contrast, all seven woodchucks transfected with wild-type WHV DNA developed markers consistent with viral infection. Thus, it is likely (P < 0.01) that the WHV X gene is important for virus replication in the natural host.
WIN 51711-dependent mutants of poliovirus type 3: evidence that virions decay after release from cells unless drug is present
Authors: Mosser A G , Rueckert R R
Published: January 2020

Twenty-two spontaneous mutants of the Sabin strain of poliovirus type 3 were selected for drug resistance by plating on HeLa cell monolayers in the presence of WIN 51711, an uncoating inhibitor. When replated in the presence and absence of drug, two classes of mutants were observed; mutants displayed either a drug-dependent or a non-drug-dependent phenotype, in the proportion 14:8. Non-drug-dependent mutants plaqued with equal efficiency in the presence or absence of drug. By contrast, drug-dependent mutants made no plaques in the absence of drug, except for revertants. In single-step growth curve experiments, however, drug-dependent mutants grew as well in the absence of drug as in its presence. This paradoxical behavior of dependent mutants was traced to extreme thermolability at 37 degrees C (12- to 30-s half-life) in the absence of drug. Thermolability was exhibited only after the virus was released from the cell, implying the presence of a cell-associated protective factor, possibly pocket factor. Thus, in the absence of a thermostabilizing drug, drug-dependent mutants decayed too rapidly after release to permit spread in the plaque assay. The thermodecay product was shown to consist of 135S particles lacking VP4.
Characterization of a glial cell line persistently infected with borna disease virus (BDV): influence of neurotrophic factors on BDV protein and RNA expression
Authors: Carbone K M , Rubin S A , Sierra-Honigmann A M , Lederman H M
Published: January 2020

Borna disease virus (BDV) infects cells of the nervous system in a wide range of species. Previous work suggests that there are differences in BDV replication in neuronal cells and glial cells. Many neurons are lysed by the immunopathologic response to BDV; lysis of dentate gyrus neurons in the absence of encephalitis is seen in rats inoculated with BDV as neonates. In contrast, persistently BDV-infected astrocytes increase over the course of BDV infection. Therefore, we compared BDV replication in neuronal (SK-N-SH and SK-N-SHEP) and astrocytic (C6) cell lines. While SK-N-SH cells produced more infectious virions per cell, the C6 cells contained more BDV proteins and RNA. BDV sequences in the supernatants of both cell types were identified, despite low titers of infectious virus, suggesting the release of incomplete virions into the medium. C6 cells secreted a factor or factors into the medium that enhanced the production of BDV proteins and RNA in other cell lines. In addition, nerve growth factor treatment produced the same enhancement. Thus, BDV replication in certain neural cells in vitro may be linked to the production of cell-specific factors which affect viral replication.
All three domains of the Epstein-Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts
Authors: Moorthy R K , Thorley-Lawson D A
Published: January 2020

LMP-1, the Epstein-Barr virus latent membrane protein 1, is the only protein encoded by the virus that has been shown to have the properties of a transforming oncogene in rodent fibroblasts such as Rat-1 cells. LMP-1 is phosphorylated and proteolytically cleaved in Rat-1 cells in a manner similar to that seen in human lymphocytes. In this study, we demonstrate that all three major domains of LMP-1 (N-terminal, transmembrane, and C-terminal domains) are required for the ability to transform Rat-1 cells in culture, as assayed by loss of contact inhibition. This study is the first demonstration of a functional role for the C-terminal domain of LMP-1. Our analysis suggests that there are at least three distinct regions of the C terminus involved in signalling. Amino acids 306 to 334, which generate a toxic signal in the absence of amino acids 334 to 364, and the last 23 amino acids, 364 to 386, are essential for transformation. Biochemical analysis of the LMP-1 mutants with the three domains deleted indicate that the mutant N-terminal with the domain deleted is phosphorylated normally but is inefficiently cleaved compared with the wild-type LMP-1. The mutant with the transmembrane domain deleted is also phosphorylated but is not cleaved, showing that phosphorylation of LMP-1 does not require membrane association. The nontransforming mutant with the C-terminal domain deleted that lacks the last 23 amino acids is phosphorylated and cleaved. Therefore, these processing events alone are insufficient to generate a transforming signal.
The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms
Authors: Davies M V , Chang H W , Jacobs B L , Kaufman R J
Published: January 2020

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.
cis-acting sequences in the Aleutian mink disease parvovirus late promoter important for transcription: comparison to the canine parvovirus and minute virus of mice
Authors: Storgaard T , Christensen J , Aasted B , Alexandersen S
Published: January 2020

We are currently investigating the regulation of transcription of the Aleutian mink disease parvovirus (ADV). ADV causes a chronic immune complex-mediated condition known as classical Aleutian disease, characterized by slow viral replication. This slow replication is an intrinsic property of ADV and distinguishes it from the more prototypic parvoviruses such as minute virus of mice (MVM) and canine parvovirus (CPV). We have previously suggested a role for the weak ADV promoters in the slow replication and thereby the absence of acute cytopathology and instead establishment of persistent ADV infection with progressive immune complex-mediated chronic lesions. In this study, we have mapped the cis-acting sequences around the ADV P36 promoter responsible for both constitutive transcription and transactivation mediated by the nonstructural protein 1. The mapping was performed by using endpoint deletions of the ADV P36 promoter and by making chimeras between the ADV P36 and MVM P38 promoters. We found the weak constitutive activity of the ADV P36 promoter to be caused by suboptimal promoter proximal sequences, while the low level of transactivation was caused mainly by an upstream region including sequences with homology to the transactivation responsive element (tar) of the H-1 parvovirus (M.-L. Gu, F.-X. Chen, and S. L. Rhode, Virology 187:10-17, 1992). We also found the corresponding regions in the MVM and CPV P38 promoters to be important for transactivation of these promoters by making 5' deletions of the promoter region. In addition, it was found that MVM tar-like and upstream sequences could transfer high nonstructural protein 1 responsiveness to the ADV promoter even though the distance between the tar-like element and the TATA box was significantly changed. On the basis of comparative data for ADV, MVM, CPV, and H-1, a new clustered motif (TTGGTT) is proposed to be the responsive cis-acting element for transactivation. Homology comparison of the specific transcriptional elements of the ADV P36, MVM P38, and CPV P38 promoters suggests that few, but crucial, changes in the ADV P36 promoter and upstream region are responsible for the weak constitutive activity and low level of transactivation of the ADV P36 promoter.
Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses
Authors: Tikoo S K , Zamb T J , Babiuk L A
Published: January 2020

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.
Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site
Authors: Chen B F , Hwang L H , Chen D S
Published: January 2020

We cloned the 5' nontranslated region (NTR) from the genome of swine vesicular disease virus (SVDV), a member of the family Picornaviridae, and used it to construct a bicistronic retroviral vector. The vector is characterized by coexpression of two genes from a single transcript. We found that inclusion of the 5' NTR of SVDV did not negate the viral vector titer. Protein analysis indicated that the 5' NTR could efficiently direct internal initiation, thus allowing the downstream gene to be translated. Translation of the internally initiated porcine growth hormone gene was about 30-fold less than that when the porcine growth hormone gene was at the upstream position in NIH 3T3 cells but was about equivalent to that in HeLa cells, implying that some cellular factors that stimulated internal initiation of the SVDV 5' NTR are present in HeLa cells. However, in G418-selected clones, the Neor-encoding gene was expressed with equivalent efficiency either at a downstream position or at an upstream position in either NIH 3T3 or HeLa cells. Compared with the conventional double-gene vector or the U3-based vector, the bicistronic vector coexpressed two genes much more efficiently, owing to elimination of promoter interference. Furthermore, this type of vector infected and expressed the target genes efficiently in two primary cell lines, rat embryo and human skin fibroblast cells, which we tested. These experimental data suggest a better design for the retroviral vector and provide evidence that internal initiation of the SVDV 5' NTR was stimulated cell specifically.
WIN 52035-2 inhibits both attachment and eclipse of human rhinovirus 14
Authors: Shepard D A , Heinz B A , Rueckert R R
Published: January 2020

WIN compounds inhibit attachment of human rhinovirus 14 by binding to a hydrophobic pocket within the capsid and inducing conformational changes in the canyon floor, the region that binds the cellular receptor. To study the basis of drug resistance, we isolated and characterized a family of human rhinovirus 14 mutants resistant to WIN 52035-2. Thermostabilization data and single-cycle growth curves provided evidence for two classes of resistant mutants. One class, here called exclusion mutants, showed a marked decrease in drug-binding affinity and was characterized by substitution to bulkier amino acid side chains at two sites lining the hydrophobic pocket. The other class, called compensation mutants, displayed single-amino-acid substitutions in the drug-deformable regions of the canyon; these mutants were able to attach to cells despite the presence of bound drug. A delay in the rise period of the growth curves of compensation mutants indicated a second locus of drug action. WIN 52035-2 was found to inhibit the first step of uncoating, release of VP4. Attempts to identify this site of drug action by using single-step growth curves were obscured by abortive elution of a major fraction of cell-attached virus. The drug had no effect on the rate of this process but did affect the spectrum of particles produced.
A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H
Authors: Roop C , Hutchinson L , Johnson D C
Published: January 2020

Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) is essential for virus entry into cells and forms a hetero-oligomer with a newly described viral glycoprotein, gL. Normal folding, posttranslational processing, and intracellular transport of both gH and gL depend upon the coexpression of gH and gL in cells infected with vaccinia virus vectors (L. Hutchinson, H. Browne, V. Wargent, N. Davis-Poynter, S. Primorac, K. Goldsmith, A. C. Minson, and D. C. Johnson, J. Virol. 66:2240-2250, 1992). Homologs of gH and gL have been found in herpesviruses of all subgroups, and thus it appears likely that the gH-gL complex serves a highly conserved function during herpesvirus penetration into cells. To examine the role of gL in the infectious cycle of HSV-1, a mutant HSV-1 unable to express gL was constructed by inserting a lacZ gene cassette into the coding sequences of the UL1 (gL) gene. Because gL was found to be essential for virus replication, cell lines capable of expressing gL were constructed to complement the virus mutant. In the absence of gL, virus particles were produced, and these particles reached the cell surface; however, gL-negative particles purified from infected cells were also deficient in gH. Mutant virions lacking gH and gL were able to adsorb onto cells but were unable to enter cells and initiate an infection. Further, the role of gL in fusion of infected cells was reexamined. A mutation in HSV-1 (804) which produces the syncytial phenotype had previously been mapped to a region of the HSV-1 genome which includes the UL1 gene and no other open reading frame. However, in contrast to this previous report, we found that the syncytial mutation in 804 affects the UL53 gene, which encodes gK, a gene commonly mutated in syncytial viruses.
A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor
Authors: Blair W S , Li X , Semler B L
Published: January 2020

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.
Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence
Authors: Broome R L , Vo P T , Ward R L , Clark H F , Greenberg H B
Published: January 2020

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.
Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus
Authors: Chang M F , Sun C Y , Chen C J , Chang S C
Published: January 2020

The functions of delta antigens (HDAgs) in the replication of hepatitis delta virus (HDV) have been identified previously. The small HDAg acts as a transactivator, whereas the large HDAg has a negative effect on replication. To understand the molecular mechanisms involved in the control of HDV replication, we have established a replication system in Huh-7 cells by cotransfecting a monomeric cDNA genome of HDV and a plasmid encoding the small HDAg. We demonstrate that a leucine repeat in the middle domain of the small HDAg is involved in binding to the HDV genome and transactivation of HDV replication. When the leucine repeat was disrupted by a substitution of valine for leucine at position 115, both RNA-binding and transactivation activity of the small HDAg were abolished. In contrast, the binding and transactivation activities were not affected when Leu-37 and Leu-44 of the small HDAg were replaced by valines. In addition, small and large HDAgs can interact with each other to form protein complexes in vitro. The complex formation that may lead to the trans-dominant negative regulation of large HDAg in HDV replication is mediated by a cryptic signal located between amino acid residues 35 and 65 other than the putative N-terminal leucine zipper motif. Furthermore, an extra 21-amino-acid extension near the N terminus converts the small HDAg into a pseudo-large HDAg with negative regulation activity of HDV replication even though the extreme C-terminal residue is unchanged.
Basic amino acids flanking the zinc finger of Moloney murine leukemia virus nucleocapsid protein NCp10 are critical for virus infectivity
Authors: Housset V , De Rocquigny H , Roques B P , Darlix J L
Published: January 2020

Nucleocapsid (NC) protein NCp10 of Moloney murine leukemia virus is encoded by the 3' domain of gag and contains a zinc finger surrounded by basic amino acids. During virion assembly, NC protein is necessary for core formation and the NC zinc finger is required for the packaging of the genomic RNA dimer. In vitro NCp10 has RNA-binding and -annealing activities critical for virus infectivity, since NCp10 promotes dimerization of viral RNA containing the Psi packaging element and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (primer-binding site). To investigate the role of the basic amino acids flanking the NCp10 zinc finger, neutral residues were substituted for the basic amino acids and the effects of these mutations in vivo on virus assembly and infectivity and in vitro on the RNA-annealing activity of NCp10 were analyzed. Here we report that the substitution of 1 or 2 neutral amino acids for the basic residues did not impair the production of mature virions but that infectivity was either moderately or strongly attenuated. When more than 2 basic residues were replaced by neutral amino acids, viruses were poorly infectious because of a severe defect in genomic RNA dimer packaging and initiation of reverse transcription. In vitro NCp10-derived peptides with similar mutations were chemically synthesized and were found to be either fully or partially active or completely inactive. These data indicate that the basic residues flanking the zinc finger of NCp10 are required for the production of infectious Moloney murine leukemia virus virions.
Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells
Authors: Jarvis D L
Published: January 2020

Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D. L. Jarvis, J. G. W. Fleming, G. R. Kovacs, M. D. Summers, and L. A. Guarino, Bio/Technology 8:950-955, 1990). These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase. Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter. The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines. The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein. These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes. Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection. The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined. However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection. Thus, the same promoter behaved quite differently late in infection, depending on its local environment. Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes. The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.