bluuebunny/crossref_metadata_2025 huggingface dataset:
Phorbol esters modulate the phosphorylation of human T-cell leukemia virus type I Tax
Authors: Fontes J D , Strawhecker J M , Bills N D , Lewis R E , Hinrichs S H
Published: January 2020

The Tax protein from human T-cell leukemia virus type I (HTLV-I) is a 40-kDa phosphoprotein capable of activating transcription from its own long terminal repeat (LTR), as well as increasing the transcription of cellular genes. Transcriptional activation of the HTLV-I LTR has been demonstrated via a cyclic-AMP-responsive element within the 21-bp Tax-responsive elements of the LTR. Phorbol esters also upregulate expression via the LTR. Since phosphorylation of Tax may play a role in these processes, we investigated the relative effects of kinase-stimulating agents on 32P incorporation into Tax. Our studies demonstrated that the phorbol ester 4 beta-phorbol-12 beta-myristate-13 alpha-acetate greatly stimulated Tax phosphorylation in a time- and dose-dependent manner. In contrast, 8-bromoadenosine 3'-5'-cyclic monophosphate induced little stimulation of Tax phosphorylation. Tax phosphorylation occurred only on serine residues and was mapped to a single tryptic fragment in both Tax-producing human lymphocytes and mouse fibroblast cells.
Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16
Authors: Apt D , Chong T , Liu Y , Bernard H U
Published: January 2020

The transcription of human papillomavirus type 16 (HPV-16) is mediated by the viral enhancer. Epithelial cell-specific activation is achieved by the cooperative interaction of apparently ubiquitous transcriptional factors. One of them, nuclear factor I (NFI), binds seven sites within the HPV-16 enhancer. Point mutations on enhancer fragments, which retain epithelial cell specificity, verify the functional contribution of NFI. In band shift experiments, the epithelial cell-derived NFI proteins CTF-1, CTF-2, and CTF-3 form a characteristic pattern of heterodimeric complexes which are observed in all epithelial cells tested. Divergence from this pattern in fibroblasts, liver cells, and lymphoid cells correlates with the lack of HPV-16 enhancer activation. The HPV-16 enhancer can be activated by CTF-1 in SL-2 cells, which lack NFI-like proteins. However, exogenous CTF-1 fails to overcome the inactivity of the viral enhancer in fibroblasts. Western immunoblot and supershift analysis shows that exogenously introduced CTF-1 proteins form different heterodimer complexes with the given subset of endogenous NFI proteins in epithelial or fibroblast cells. Polymerase chain reaction analysis and cDNA library screens identified the endogenous fibroblast type NFI as NFI-X, an NFI family member originally cloned from hamster liver cells. The strict correlation between the activation or lack of activation of the HPV-16 enhancer and cell-specific subsets of NFI proteins argues for the pivotal role of NFI binding sites in the epithelial cell-specific function of the viral enhancer.
Entry of poliovirus into cells does not require a low-pH step
Authors: Pérez L , Carrasco L
Published: January 2020

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.
The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly
Authors: Kjems J , Sharp P A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev binds to a cis-acting Rev-responsive element (RRE) located within the env region of HIV-1. It has previously been shown that a 17-amino-acid peptide, corresponding to the basic domain of Rev, specifically inhibited in vitro the splicing of mRNAs containing the RRE. In this reaction, the peptide acts after an ATP-dependent step in the spliceosome assembly resulting in an accumulation of a 45-50S splicing-deficient complex. Characterization of this complex revealed that the basic domain of Rev does not interfere with U1 small nuclear ribonucleoprotein binding but blocks the entry of U4, U5, and U6 small nuclear RNAs into the spliceosome. Binding of U2 small nuclear ribonucleoprotein was partially inhibited. The critical nature of the oligomeric structure of RRE has been investigated both in vitro and in vivo. Reporter genes that contained one, three, or six repeated-monomer high-affinity Rev binding sites (IIB) within an intron yielded a correlation among the oligomeric state of bound Rev; inhibition of splicing; ability to block the assembly of U4, U5, and U6 small nuclear RNAs in the spliceosome in vitro; and level of Rev response in vivo.
Immunochemical analysis of the gp120 surface glycoprotein of human immunodeficiency virus type 1: probing the structure of the C4 and V4 domains and the interaction of the C4 domain with the V3 loop
Authors: Moore J P , Thali M , Jameson B A , Vignaux F , Lewis G K , Poon S W , Charles M , Fung M S , Sun B , Durda P J
Published: January 2020

We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gp120 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gp120 sequence, (E/K)VGKAMYAPP, in C4 were differentially sensitive to denaturation of gp120, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gp120 mutant with an alteration in the sequence of the V3 loop, and their binding to gp120 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gp120, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in an around the C4 region.
The role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly
Authors: Black B L , Rhodes R B , McKenzie M , Lyles D S
Published: January 2020

Recently, the vesicular stomatitis virus matrix (M) protein has been shown to be capable of inhibition of host cell-directed transcription in the absence of other viral components (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). M protein is a major structural protein that is known to play a critical role in virus assembly by binding the helical ribonucleoprotein core of the virus to the cytoplasmic surface of the cell plasma membrane during budding. In this study, two M protein mutants were tested to determine whether the inhibition of host transcription by M protein is an indirect effect of its function in virus assembly or whether it represents an independent function of M protein. The mutant M protein of the conditionally temperature-sensitive (ts) vesicular stomatitis virus mutant, tsO82, was found to be defective in its ability to inhibit host-directed gene expression, as shown by its inability to inhibit expression of a cotransfected target gene encoding chloramphenicol acetyltransferase. The ability of the tsO82 M protein to function in virus assembly was similar to that of wild-type M protein, as shown by its ability to complement the group III ts M protein mutant, tsO23. Another mutant, MN1, which lacks amino acids 4 to 21 of M protein demonstrated that the abilities of M protein to inhibit chloramphenicol acetyltransferase gene expression and to localize to the nucleus were unaffected by deletion of this lysine-rich amino-terminal region but that the ability to function in virus assembly was ablated. Thus, the two M protein mutants examined in this study exhibited complementary phenotypes: tsO82 M protein functioned in virus assembly but was defective in inhibition of host-directed gene expression, while MN1 M protein functioned in inhibiting gene expression but was unable to function in virus assembly. These data demonstrate that the role of M protein in inhibition of host transcription can be separated genetically from its role in virus assembly.
Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus
Authors: Bogerd H P , Fridell R A , Blair W S , Cullen B R
Published: January 2020

The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.
Isolation and characterization of new wild-type isolates of bovine lentivirus
Authors: Suarez D L , VanDerMaaten M J , Wood C , Whetstone C A
Published: January 2020

Two new isolates of bovine lentivirus, also known as bovine immunodeficiency-like virus (BIV), were obtained from a seropositive cattle herd in Florida. This is the first report of new isolates of BIV since the original BIV strain, R29, was isolated in 1969. The two new BIV isolates were derived from blood buffy coat cells cocultivated in vitro with fetal bovine lung cell cultures. The new isolates differed in vitro from the original R29 isolate in replication and syncytium formation in fetal bovine lung cells. Both new isolates were confirmed as BIV by immunofluorescence assay, Western blotting (immunoblotting), and polymerase chain reaction. Sequence analyses of the polymerase chain reaction pol gene product showed 92.6 and 93.6% homology to the published nucleotide sequence of BIV R29-127, a molecular clone derived from BIV R29. Each of the new BIV isolates was inoculated into two calves, and virus was recovered between 5 and 10 days postinoculation (p.i.), with BIV seroconversion between 10 and 21 days p.i. Virus was recoverable and antibody was detectable for at least 4 months p.i. Two calves developed a transiently elevated mononuclear cell count, similar to what was reported for BIV R29 in the original experimental calf inoculations. No other clinical abnormalities were observed.
Deletion of DNA encoding the first five transmembrane domains of Epstein-Barr virus latent membrane proteins 2A and 2B
Authors: Longnecker R , Miller C L , Tomkinson B , Miao X Q , Kieff E
Published: January 2020

A recombinant Epstein-Barr virus (EBV) was constructed, with a positive-selection marker inserted at the site of a deletion of a DNA segment which encodes the first five transmembrane domains of LMP2A and LMP2B. Despite the mutation, the mutant recombinant EBV was able to initiate and maintain primary B-lymphocyte growth transformation in vitro. Cells transformed with the mutant recombinant were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, or serum requirement. Expression of EBNA1, EBNA2, EBNA3A, EBNA3C, and LMP1 and permissivity for lytic EBV infection were also unaffected by the LMP2 deletion mutation. These results complete the molecular genetic studies proving LMP2 is dispensable for primary B-lymphocyte growth transformation, latent infection, and lytic virus replication in vitro.
Pathogenesis of murine gammaherpesvirus infection in mice deficient in CD4 and CD8 T cells
Authors: Ehtisham S , Sunil-Chandra N P , Nash A A
Published: January 2020

Murine gammaherpesvirus is a natural pathogen of wild mice. The virus infects alveolar cells and spleen cells during the primary infection and establishes a latent/persistent infection in B lymphocytes. Little is known about the immunological response to gammaherpesviruses during a primary infection. To address this issue, we investigated the pathogenesis of murine herpesvirus 68 (MHV-68) infection in mice deficient in CD4 or CD8 T-cell populations. Infection of the lung and spleen were greatly exacerbated in CD8-deficient mice, reflected by elevated virus titers in the lung and an increase in the number of infected splenocytes located around germinal centers. This finding contrasts with clearance of virus from the lung and spleen by day 12 postinfection in CD4-depleted animals. These data clearly indicate a major role for CD8 T cells in recovery from an acute MHV-68 infection. Whereas CD4 T cells fail to influence the course of infection in the lung, they do contribute to lymphoproliferation seen in the spleen (splenomegaly) during the primary infection. The significance of these results are discussed in relation to the immune response to other herpesviruses, in particular Epstein-Barr virus, with which MHV-68 shares similar molecular and biological properties.
Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain
Authors: Cohen B D , Goldstein D J , Rutledge L , Vass W C , Lowy D R , Schlegel R , Schiller J T
Published: January 2020

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex.
The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium
Authors: Martinez-Pomares L , Stern R J , Moyer R W
Published: January 2020

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.
Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block
Authors: Wang C , Takeuchi K , Pinto L H , Lamb R A
Published: January 2020

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.
Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1
Authors: Lu Y Y , Koga Y , Tanaka K , Sasaki M , Kimura G , Nomoto K
Published: January 2020

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.
Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus
Authors: Lewis P F , Emerman M
Published: January 2020

The human immunodeficiency virus productively infects and integrates into cells that have been arrested in the cell cycle with either gamma irradiation or aphidicolin. Integration by oncoretroviruses such as the murine leukemia virus (MuLV), on the other hand, depends on cell proliferation. Although the entire cell cycle is not necessary for MuLV infection, it is essential that the infected cells pass through mitosis. The long terminal repeat circle junction, a marker for nuclear entry, is first observed in MuLV-infected cells immediately after mitosis. These results suggest that mitosis is necessary for nuclear entry of MuLV, but not human immunodeficiency virus, unintegrated proviral DNA.
Retention of the herpes simplex virus type 1 (HSV-1) UL37 protein on single-stranded DNA columns requires the HSV-1 ICP8 protein
Authors: Shelton L S , Albright A G , Ruyechan W T , Jenkins F J
Published: January 2020

The UL37 and ICP8 proteins present in herpes simplex virus type 1 (HSV-1)-infected-cell extracts produced at 24 h postinfection coeluted from single-stranded-DNA-cellulose columns. Experiments carried out with the UL37 protein expressed by a vaccinia virus recombinant (V37) revealed that the UL37 protein did not exhibit DNA-binding activity in the absence of other HSV proteins. Analysis of extracts derived from cells coinfected with V37 and an ICP8-expressing vaccinia virus recombinant (V8) and analysis of extracts prepared from cells infected with the HSV-1 ICP8 deletion mutants d21 and n10 revealed that the retention of the UL37 protein on single-stranded DNA columns required a DNA-binding-competent ICP8 protein.
Sequence conservation in field and experimental isolates of Borna disease virus
Authors: Schneider P A , Briese T , Zimmermann W , Ludwig H , Lipkin W I
Published: January 2020

Coding and noncoding sequences were analyzed from field and experimental isolates of Borna disease virus. For a 24-kDa protein, maximum divergence was 1.5% at the predicted amino acid level and 3.1% at the nucleotide level. For a 40-kDa protein, maximum divergence was 1.1% at the predicted amino acid level and 4.1% at the nucleotide level. The highest variability in sequence (10%) was found in a 40-nucleotide stretch of genomic RNA between coding sequences for the 40- and 24-kDa proteins. The degree of sequence conservation in these isolates, passaged in various host species in vivo and in vitro over a period of 64 years, is unusual for negative-strand RNA viruses.
Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function
Authors: Maisner A , Schneider-Schaulies J , Liszewski M K , Atkinson J P , Herrler G
Published: January 2020

Two cellular proteins, membrane cofactor protein (MCP) and moesin, were reported recently to be functionally associated with the initiation of a measles virus infection. We have analyzed the interaction of measles virus with cell surface proteins, using an overlay binding assay with cellular proteins immobilized on nitrocellulose. Among surface-biotinylated proteins from a human rectal tumor cell line (HRT), measles virus was able to bind only to a 67-kDa protein that was identified as MCP. The virus recognized different isoforms of MCP expressed from human (HRT and HeLa) and simian (Vero) cell lines. The binding of measles virus to MCP was abolished after cleavage of the disulfide bonds by reducing agents as well as after enzymatic release of N-linked oligosaccharides. By contrast, removal of sialic acid or O-linked oligosaccharides did not affect the recognition of MCP measles virus. These data indicate that the receptor determinant of MCP is dependent on a conformation of the protein that is maintained by disulfide bonds and N-glycans present in the complement binding domains. Our results are consistent with a role of MCP as primary attachment site for measles virus in the initial stage of an infection. The functional relationship between MCP and moesin in a measles virus infection is discussed.
Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis
Authors: Hellen C U , Pestova T V , Wimmer E
Published: January 2020

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.
Induction of antibody responses that neutralize human T-cell leukemia virus type I infection in vitro and in vivo by peptide immunization
Authors: Tanaka Y , Tanaka R , Terada E , Koyanagi Y , Miyano-Kurosaki N , Yamamoto N , Baba E , Nakamura M , Shida H
Published: January 2020

In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pep190-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pep180-204, but not pep185-194 or pep194-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 10(7) live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pep190-199- and pep180-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo.
Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network
Authors: Gershon A A , Sherman D L , Zhu Z , Gabel C A , Ambron R T , Gershon M D
Published: January 2020

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.
Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA
Authors: Wang Y F , Sawicki S G , Sawicki D L
Published: January 2020

The alphavirus mutant Sindbis virus HR ts4, which has been assigned to the A complementation group, possessed a selective defect in negative-strand synthesis that was similar although not identical to that observed for the B complementation group mutant ts11 (Y.-F. Wang, S. G. Sawicki, and D. L. Sawicki, J. Virol. 65:985-988, 1991). The causal mutation was identified as a change of a C to a U residue at nucleotide 4903 in the nsP3 open reading frame that predicted a change of Ala-268 to Val. Thus, both nsP3 and nsP1 play a role selectively in the transcription of negative strands early in infection. The assignment of the mutation carried by an A complementation group mutant of Sindbis virus HR to nsP3 was unexpected, as mutations in other A complementation group mutants studied to date mapped to nsP2. Another mutant with a conditionally lethal mutation, ts7 of the G complementation group, also possessed a causal mutation resulting from a single-residue change in nsP3. Negative-strand synthesis ceased more slowly after a shift to the nonpermissive temperature in ts7-than in ts4-infected cells, and ts7 complemented ts11, but ts4 did not. However, the nsP3 of both ts4 and ts7 allowed reactivation of negative-strand synthesis by stable replication complexes containing nsP4 from ts24. Therefore, mutations in nsP3 affected only early events in replication and probably prevent the formation and/or function of the initial replication complex that synthesizes its negative-strand template. Because neither ts4 nor ts7 complemented 10A complementation group mutants, the genes for nsP2 and nsP3 function initially as a single cistron. We interpret these findings and present a model to suggest that the initial alphavirus replication complex is formed from tightly associated nsP2 and nsP3, perhaps in the form of P23, and proteolytically processed and trans-active nsP4 and nsP1.
Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus
Authors: Boniotti B , Wirblich C , Sibilia M , Meyers G , Thiel H J , Rossi C
Published: January 2020

Expression studies conducted in vitro and in Escherichia coli led to the identification of a protease from rabbit hemorrhagic disease virus (RHDV). The gene coding for this protease was found to be located in the central part of the genome preceding the putative RNA polymerase gene. It was demonstrated that the protease specifically cuts RHDV polyprotein substrates both in cis and in trans. Site-directed mutagenesis experiments revealed that the RHDV protease closely resembles the 3C proteases of picornaviruses with respect to the amino acids directly involved in the catalytic activity as well as to the role played by histidine as part of the substrate binding pocket.
An assembly domain of the Rous sarcoma virus Gag protein required late in budding
Authors: Wills J W , Cameron C E , Wilson C B , Xiang Y , Bennett R P , Leis J
Published: January 2020

The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding.
Protective antibodies inhibit reovirus internalization and uncoating by intracellular proteases
Authors: Virgin H W , Mann M A , Tyler K L
Published: January 2020

We identified in vitro correlates of in vivo protection mediated by nonneutralizing antibodies specific for reovirus capsid proteins. We defined mechanisms of antibody action by analyzing monoclonal antibody (MAb) effects at sequential steps in reovirus serotype 3 strain Dearing (T3D) infection of L cells. Two types of experiments showed that protective MAbs specific for the outer capsid proteins sigma 3 or mu 1 inhibited T3D infection independent of effects on binding. First, MAbs which had no effect on T3D binding inhibited T3D growth. Second, MAb-coated T3D attached to L cells did not replicate as efficiently as T3D without bound antibody. We therefore defined sigma 3-specific MAb effects on postbinding steps in T3D infection. T3D coated with MAb sigma 3-10G10 exhibited prolonged sensitivity to growth inhibition by ammonium chloride. Since ammonium chloride inhibits endosomal acidification and proteolytic processing of the T3D capsid, this suggested that MAbs inhibit early steps in T3D infection. This was confirmed by direct demonstration that several sigma 3-specific MAbs inhibited proteolytic uncoating of virions by fibroblasts. We identified two mechanisms for antibody-mediated inhibition of virion uncoating: (i) inhibition of internalization of T3D-MAb complexes bound to the cell surface, and (ii) inhibition of intracellular proteolysis of the T3D capsid. Studies using a cell-free system confirmed that sigma 3-specific MAbs directly block proteolytic uncoating of the T3D virion. In addition, we found that sigma 3-specific MAbs block (and therefore define) two distinct steps in proteolytic uncoating of the reovirion. We conclude that antibodies which are protective in vivo inhibit postbinding events in reovirus infection of permissive cells. Protective antibodies act by inhibiting internalization and intracellular proteolytic uncoating of the virion. Analysis of postbinding mechanisms of MAb action may identify targets for vaccine development and antiviral therapy.
In murine AIDS, B cells are early targets of defective virus and are required for efficient infection and expression of defective virus in T cells and macrophages
Authors: Kim W K , Tang Y , Kenny J J , Longo D L , Morse H C
Published: January 2020

Previous studies showed that B cells and CD4+ T cells are required for induction of a murine retrovirus-induced immunodeficiency syndrome, murine AIDS. Using B6 mice deficient in mature B cells as a result of a knockout of the transmembrane exon of the immunoglobulin M gene, we found that spleen and other tissues from murine AIDS virus-infected mice did not express the defective virus (BM5def) required for induction of disease, even though helper viruses were readily detectable and BM5def proviral DNA was present. This indicates that the B-lineage cells are the primary targets for infection and expression of the defective virus and that in the absence of mature B cells, there is inefficient infection of T cells and macrophages.
The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation
Authors: Belli B , Fan H
Published: January 2020

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.
Selective inhibition of virus protein synthesis by prostaglandin A1: a translational block associated with HSP70 synthesis
Authors: Amici C , Giorgi C , Rossi A , Santoro M G
Published: January 2020

Cyclopentenone prostaglandins are potent inhibitors of virus replication. The antiviral activity has been associated with the induction of 70-kDa heat shock protein (HSP70) synthesis. In this report, we describe that in African green monkey kidney cells infected with Sendai virus (SV) and treated with prostaglandin A1 (PGA1), SV protein synthesis was selectively blocked as long as HSP70 was being synthesized by the host cell. The block appeared to be at the translational level, as indicated by the following (i) PGA1 had no effect on SV primary transcription, and a dramatic decrease in the abundance of SV mRNA occurred only at later stages of infection; and (ii) treatment with PGA1 started at 6 h postinfection, at which time SV mRNA had already accumulated in infected cells, did not suppress the levels of NP mRNA, but it reduced the amount of ribosome-bound NP mRNA and caused a dramatic decrease in the level of genomic RNA. The PGA1-induced block of SV protein synthesis appeared to be cell mediated, since it was prevented by actinomycin D, while PGA1 had no effect on SV mRNA translation in vitro. The possibility that HSP70 could be a mediator of the antiviral effect is suggested by the fact that treatment with other classical inducers of HSP70, including sodium arsenite, cadmium, and heat shock at 42 degrees C for 5 h, also selectively prevented SV protein synthesis as long as heat shock protein synthesis occurred. Moreover, SV protein synthesis was not inhibited by PGA1 in murine Friend erythroleukemic cells, which lack the ability to induce HSP70 expression in response to PGA1.
Mutations in the cytoplasmic tail of herpes simplex virus glycoprotein H suppress cell fusion by a syncytial strain
Authors: Wilson D W , Davis-Poynter N , Minson A C
Published: January 2020

We have developed a complementation assay, using transiently transfected COS cells, to facilitate a molecular analysis of the herpes simplex virus type 1 glycoprotein gH. When infected by a gH-null syncytial virus, COS cells expressing wild-type gH generate infectious progeny virions and form a syncytium with neighboring cells. By deletion and point mutagenesis, we have found particular residues in the gH cytoplasmic tail to be essential for generation of a syncytium but apparently dispensable for production of infectious virions. This study emphasizes the different requirements for cell-cell and cell-envelope fusion and demonstrates that changes in the non-syn locus UL22-gH can reverse the syncytial phenotype.
A small yeast RNA selectively inhibits internal initiation of translation programmed by poliovirus RNA: specific interaction with cellular proteins that bind to the viral 5'-untranslated region
Authors: Das S , Coward P , Dasgupta A
Published: January 2020

We have purified, sequenced, and prepared a synthetic clone of a small (60-nucleotide) RNA molecule from the yeast Saccharomyces cerevisiae that had previously been isolated on the basis of its ability to selectively block the translation of poliovirus mRNA. RNA derived from the clone by transcription with T7 RNA polymerase appears to block translation initiation by internal ribosome entry (cap independent) but does not significantly affect cap-dependent translation. Deletion analysis of the poliovirus 5'-untranslated region (5'-UTR) has shown that yeast inhibitor RNA (I-RNA) requires internal ribosome entry site sequences to inhibit the translation of poliovirus RNA in vitro. Using a bicistronic RNA construct, we show that I-RNA preferentially inhibits translation by internal ribosome entry. Gel retardation and UV cross-linking studies demonstrate that I-RNA specifically binds proteins which interact with RNA secondary structures within the poliovirus 5'-UTR presumably involved in internal initiation. Specifically, purified I-RNA competes with virus RNA structures within the 5'-UTR which bind a cellular protein with an approximate molecular mass of 52 kDa. Finally, when transfected into HeLa cells, I-RNA efficiently inhibits the replication of poliovirus RNA presumably by inhibiting translation of the input virus RNA.
High incidence of HAM/TSP-like symptoms in WKA rats after administration of human T-cell leukemia virus type 1-producing cells
Authors: Kushida S , Mizusawa H , Matsumura M , Tanaka H , Ami Y , Hori M , Yagami K , Kameyama T , Tanaka Y , Yoshida A
Published: January 2020

We demonstrate a significantly high incidence of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM)-or tropical spastic paraparesis (TSP)-like symptoms in WKA rats after injection with HTLV-1-producing MT-2 cells, while no symptoms were observed in F344 rats injected with MT-2 cells or in control WKA rats. Five of the eight (63%) WKA rats injected with MT-2 cells showed HAM/TSP-like paraparesis at 105 weeks of age, but none of seven MT-2-injected F344 rats or eight control WKA rats showed symptoms. This high incidence of HAM/TSP-like symptoms in WKA rats was statistically significant (P < 0.05). Six of the eight (75%) WKA rats injected with MT-2 cells showed HAM/TSP-like paraparesis at 108 weeks of age. HAM/TSP-like symptoms were also observed in one of the two WKA rats injected with HTLV-1-producing Ra-1 cells at 128 weeks of age. HTLV-1 provirus was detected in peripheral blood mononuclear cells in both WKA and F344 rats. The provirus was detected in the spinal cords of the HAM/TSP-like WKA rats that had severe neuropathological changes. WKA and F344 rats showed no significant difference in antibody response against HTLV-1 Gag antigen. However, the antibody response against the C-terminal half of gp46 HTLV-1 envelope protein was lower in WKA rats than in F344 rats. Pathological analysis of the HAM/TSP-like rats showed degeneration of the white matter of the spinal cord and peripheral nerves. These findings suggest that both the genetic background of the host and HTLV-1 infection are important in neuropathogenesis of HAM/TSP-like paraparesis in rats.
Effect of CD4 gene expression on adenovirus replication
Authors: Hotta J , Shi L , Ginsberg H S
Published: January 2020

The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.