bluuebunny/crossref_metadata_2025 huggingface dataset:
Minute virus of mice infection modifies cellular transcription elongation
Authors: Krauskopf A , Ben-Asher E , Aloni Y
Published: January 2020

Our previous observations indicated that upon infection with minute virus of mice (MVM), Ehrlich ascites cells lose a transcription elongation activity which is essential for the readthrough of the MVM attenuator. This was monitored by the ability of extracts from uninfected but not from infected cells to support readthrough of the P4 attenuator when added to partially purified transcription elongation complexes. We have investigated the nature of this change in transcription elongation following MVM infection. In this communication, we show that infection of Ehrlich ascites cells with MVM leads to a general shift in the length of nascent mRNA synthesized in isolated nuclei and separated by sucrose gradients. Furthermore, infection leads to attenuation of transcription of the cellular gene c-fos but not c-myc. We show biochemical evidence to support a model by which, following MVM infection, there is a functional reduction in the activity of a TFIIS-like general transcriptional elongation activity.
Spontaneous fusions to prv43 can suppress the export defect of pseudorabies virus gIII signal peptide mutants
Authors: Powers L , Ryan P
Published: January 2020

We have devised an enrichment scheme for the isolation of export-competent derivatives of pseudorabies virus glycoprotein gIII signal peptide mutants. Enrichment is based upon a growth advantage imparted upon gIII-containing virions compared with virions lacking the glycoprotein. Each of identified derivatives suppressed the gIII signal peptide defect by fusing the gIII gene in frame to the prv43 gene that lay immediately upstream; the result was the synthesis of a Prv43-gIII hybrid protein. The deduced Prv43 protein is predicted to span a membrane multiple times, and it appeared that the gIII portion of each hybrid used a hydrophobic domain of Prv43 protein to initiate its export. For at least two of the isolates, the hybrid protein was efficiently translocated across the endoplasmic reticulum membrane but appeared to be poorly exported out of the endoplasmic reticulum. Nonetheless, the prv43-gIII fusions encoded a gIII species that was localized to the virus envelope. Because the gIII portion of each hybrid protein must be exposed on the virion surface to provide a growth advantage, our results also suggest a preliminary membrane topology for wild-type Prv43 protein.
Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses?
Authors: Karlin S , Doerfler W , Cardon L R
Published: January 2020

Dinucleotide over- and underrepresentation is evaluated in all available completely sequenced DNA or RNA viral genomes, ranging in size from 3 to 250 kb (available RNA viruses fall into the small-virus category). The dinucleotide CpG is statistically underrepresented (suppressed) in all but four of the small viruses (more than 75 with lengths of < 30 kb) but has normal relative abundances in most large viruses (> or = 30 kb). Most retrotransposons in eukaryotic species also show low CpG relative abundances. Interpretations, especially in some cases of DNA viruses or viruses with a DNA intermediate, might relate to methylation effects and modes of viral integration and excision. Other possible contributing factors relate to dinucleotide stacking energies, special mutation mechanisms, and evolutionary events.
Papillomavirus contains cis-acting sequences that can suppress but not regulate origins of DNA replication
Authors: Nallaseth F S , DePamphilis M L
Published: January 2020

Bovine papillomavirus (BPV) DNA has been reported to restrict its own replication and that of the lytic simian virus 40 (SV40) origin to one initiation event per molecule per S phase, which suggests BPV DNA replication as a model for cellular chromosome replication. Suppression of the SV40 origin required two cis-acting BPV sequences (NCOR-1 and -2) and one trans-acting BPV protein. The results presented in this paper confirm the presence of two NCOR sequences in the BPV genome that can suppress polyomavirus (PyV) as well as SV40 origin-dependent DNA replication as much as 40-fold. However, in contrast to results of previous studies on SV40, most of the suppression of the PyV origin was due to NCOR-1, a 512-bp sequence that functioned independently of distance or orientation with respect to the PyV origin and that was not required for BPV DNA replication. Moreover, NCOR-1 alone or together with NCOR-2 did not restrict the ability of the PyV ori to reinitiate replication within a single S phase and did not require any BPV protein to exert suppression. Furthermore, NCOR-1 did not suppress BPV origin-dependent DNA replication except in the presence of PyV large tumor antigen (T-ag). Since NCOR-1 suppression of PyV origin activity also varied with T-ag concentration, suppression of origins by NCOR sequences appeared to require papovavirus T-ag. Therefore, it is unlikely that NCOR sequences are involved in regulating BPV DNA replication. When these results are taken together with those from other laboratories, BPV appears to be a slowly replicating version of papovaviruses rather than a model for origins of DNA replication in eukaryotic cell chromosomes.
Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random
Authors: Ewing C , Allan W , Daly K , Hou S , Cole G A , Doherty P C , Blackman M A
Published: January 2020

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.
Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly
Authors: Spearman P , Wang J J , Vander Heyden N , Ratner L
Published: January 2020

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.
Recognition of envelope protein by dengue virus serotype-specific human CD4+ CD8- cytotoxic T-cell clones
Authors: Livingston P G , Kurane I , Lai C J , Bray M , Ennis F A
Published: January 2020

We analyzed dengue virus-specific CD4+ CD8- cytotoxic T lymphocytes (CTL) at the clonal level to further understand their role in dengue virus infections. Stimulation of peripheral blood mononuclear cells from two dengue virus type 4 (D4V)-immune donors with live D4V or noninfectious D4V antigen generated 17 HLA class II-restricted CD4+ CTL capable of specific lysis of dengue virus antigen-treated autologous lymphoblastoid cell lines. Thirteen clones were D4V specific, three clones were cross-reactive for D2V and D4V, and one clone was cross-reactive for D1V, D3V, and D4V. Antigen recognition by six D4V-specific clones and three D2V- and D4V-cross-reactive clones was restricted by HLA-DR7. Five D4V-specific CD4+ CTL clones lysed autologous lymphoblastoid cell lines infected with a dengue virus-vaccinia virus recombinant containing the E gene of D4V, whereas three serotype-cross-reactive CTL clones did not. These results indicate that E-specific clones are serotype specific and HLA-DR7 restricted in these two donors and suggest that a common epitope on E protein may be recognized. E protein-specific CD4+ CTL may be important mediators of virus clearance especially during reinfection with the same serotype as that in primary infection by providing help for virus-specific antibody production and lysis of virus-infected cells.
Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes
Authors: Counter C M , Botelho F M , Wang P , Harley C B , Bacchetti S
Published: January 2020

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.
Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells
Authors: Petti L , DiMaio D
Published: January 2020

The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts.
Apoptosis: a mechanism of cell killing by influenza A and B viruses
Authors: Hinshaw V S , Olsen C W , Dybdahl-Sissoko N , Evans D
Published: January 2020

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.
The "V3" domain is a determinant of simian immunodeficiency virus cell tropism
Authors: Kirchhoff F , Mori K , Desrosiers R C
Published: January 2020

Thirty-one different mutant forms of simian immunodeficiency virus (SIVmac) were created with changes in the region of env corresponding to the V3 domain of HIV-1. Sixteen of these mutants had one amino acid change, 12 had two changes, two had three changes, and one had four changes in the SIVmac "V3" loop. The ability of the mutant viruses to replicate in CEMx174 cells, rhesus monkey peripheral blood mononuclear cells, and rhesus monkey alveolar macrophages was investigated. Ten of the mutant viruses replicated with approximately wild-type kinetics in all three cell types. Of the 31 mutants, 22 were able to replicate in one or more of the cell types. Thus, this region of SIVmac gp120 is quite tolerant to change. Nine of the mutants replicated poorly or not at all in any of the cells tested. The lack of replication competence of some of the mutants was associated with inefficient proteolytic processing of the gp160 precursor. Some mutations had dramatic differential effects in different cell types. For example, changing P to S at position 321 and M to I at position 325 drastically reduced replication in macrophages and CEMx174 cells but had no effect on replication in peripheral blood mononuclear cells. Mutants with altered tropism were blocked at an early stage that includes virus entry into cells. Thus, sequences in SIVmac that correspond to V3 in HIV-1 can affect virus entry and cell tropism in a manner analogous to that of HIV-1 V3.
Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity
Authors: Klinedinst D K , Challberg M D
Published: January 2020

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.
Sendai virus pneumonia: evidence for the early recruitment of gamma delta T cells during the disease course
Authors: Ogasawara T , Emoto M , Kiyotani K , Shimokata K , Yoshida T , Nagai Y , Yoshikai Y
Published: January 2020

We previously reported that gamma delta T cells appeared and could play a protective role early in infections with intracellular bacteria such as Listeria monocytogenes, Mycobacterium bovis BCG, and Salmonella choleraesuis. To extend these findings to virus infection, we examined the developmental sequence of gamma delta T cells in bronchoalveolar lavage during the course of Sendai virus infection in C57BL/6 mice. To produce a natural but nonlethal infection course as far as possible, we used a sublethal dose of a wild-type virus which had not been subjected to serial passages in a chicken embryo, hence retaining full virulence for mice. Virus titers in lungs reached a peak on day 6 and then decreased to an undetectable level by day 10. This time course of virus reproduction was immediately and coincidentally followed by the developmental course of gamma delta T cells, in which the cell number peaked on day 7 and then decreased to a marginal level by day 10. On the other hand, the alpha beta T-cell number continued to increase until day 10 and remained at a high level thereafter. The early-appearing gamma delta T cells were CD4-, CD8-, IL-2R alpha- beta+, CD44+, Mel-14-, and LFA-1 alpha/beta+ in phenotype and used V gamma 1/2 and V gamma 4 and V delta 3, V delta 4, V delta 5, and V delta 6. The gamma delta T cells were responding to macrophages from infected mice when the cells were cultured in vitro. Furthermore, the expression of endogenous heat shock protein (hsp) was infection specific, and its level appeared to correlate with the gamma delta T-cell development. These results suggest that the early recruitment of gamma delta T cells, which proliferate in response to endogenous hsp+ cells, is also characteristic of this virus infection, although this view appears to be contradictory to earlier reports.
Cell-binding domain of adenovirus serotype 2 fiber
Authors: Louis N , Fender P , Barge A , Kitts P , Chroboczek J
Published: January 2020

The adenovirus fiber appears as a long, thin projection terminated by a knob (head). The fiber consists of a trimeric protein whose head domain is thought to interact with cell receptors. The head part (amino acids 388 to 582) of adenovirus type 2 fiber was produced in a baculovirus expression system. The purified protein was shown to cross-link into trimers. It was very resistant to proteolytic attack and seemed to attain a high degree of compactness. The head domain efficiently inhibited attachment of adenovirus to receptors on the surface of HeLa cells, thereby confirming the hypothesis that the head domain interacts with viral receptors.
Alphaherpesvirus origin-binding protein homolog encoded by human herpesvirus 6B, a betaherpesvirus, binds to nucleotide sequences that are similar to ori regions of alphaherpesviruses
Authors: Inoue N , Dambaugh T R , Rapp J C , Pellett P E
Published: January 2020

We previously identified a human herpesvirus 6B (HHV-6B) homolog of the alphaherpesvirus origin-binding protein (OBP), exemplified by the herpes simplex virus type 1 UL9 gene product. This finding is of particular interest because HHV-6B is otherwise more closely related to members of the betaherpesvirus subfamily. The prototypic betaherpesvirus, human cytomegalovirus, does not encode an obvious OBP homolog and contains a more complex origin of replication than do alphaherpesviruses. Thus, analysis of the function of the HHV-6B OBP homolog is essential for understanding the mechanism of HHV-6B DNA replication initiation. The HHV-6B OBP homolog, OBPH6B, was expressed in vitro by coupled transcription and translation and in insect cells by infection with recombinant baculoviruses. The expressed protein bound to two DNA sequences located upstream of the HHV-6B major DNA-binding protein gene homolog, within a region that was predicted to serve as an origin of replication on the basis of its sequence properties. The binding sites lie within 23-bp segments and are similar to OBP-binding sites of herpes simplex virus type 1. The two OBPH6B-binding sequences are separated by an AT-rich region and have an imperfect dyad symmetry as do the alphaherpesvirus origin regions. We identified OBPH6B transcripts by reverse transcription PCR in HHV-6B-infected Molt-3 cells. These results suggest that OBPH6B functions in a manner analogous to the alphaherpesvirus OBP and that initiation of HHV-6B DNA replication may resemble that of alphaherpesviruses.
The Cys-His motif of Ty3 NC can be contributed by Gag3 or Gag3-Pol3 polyproteins
Authors: Orlinsky K J , Sandmeyer S B
Published: January 2020

The major structural proteins capsid and nucleocapsid (NC) of the Saccharomyces cerevisiae retroviruslike element Ty3 are produced as domains within the Gag3 and Gag3-Pol3 precursor polyproteins. Ty3 NC contains one copy of the conserved motif CX2CX4HX4C found in most retroviral NC proteins. We show here that NC proteins derived by processing of these different precursor species differ at their carboxyl termini. To determine whether the Cys-His motifs of these nascent NC domains contribute differently to replication, Gag3 and Gag3-Pol3 fusion proteins containing wild-type or mutant Cys-His domains were expressed from separate constructs. Although the Cys-His box was shown to be essential for polyprotein processing of a wild-type Ty3 element, this domain could be contributed from Gag3 or as part of Gag3-Pol3. These data suggest that the functions of the retroviral NC Cys-His domain contributed from Gag and Gag-Pol are redundant.
High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication
Authors: Burns D P , Temin H M
Published: January 2020

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.
Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis
Authors: Vanin E F , Kaloss M , Broscius C , Nienhuis A W
Published: January 2020

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution.
Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation
Authors: Tsui S , Schubach W H
Published: January 2020

Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been shown to be indispensable for immortalization of latently infected B lymphocytes, and it has been shown that EBNA-2 exists in a high-molecular-weight complex in these cells. In order to study the components of this protein machinery, we have purified baculovirus-expressed EBNA-2 from insect cells to greater than 95% homogeneity. We have shown by both gel filtration and sucrose gradient analysis that the purified material corresponds to a multimer containing eight EBNA-2 subunits. This multimeric complex is stable in 1.0 M NaCl, suggesting that the self-association is quite strong in vitro. By expressing portions of the EBNA-2 open reading frame to generate fusion proteins in yeast cells, we have used the two-hybrid system to demonstrate that this self-association occurs in vivo and is mediated at least in part by a domain of EBNA-2 encompassing amino acids 122 to 344. Mutational analysis of the self-association function suggests that two subdomains that flank amino acid 232 may each play a role in EBNA-2 protein-protein interaction.
Priming of immature thymocytes to CD3-mediated apoptosis by infection with murine cytomegalovirus
Authors: Koga Y , Tanaka K , Lu Y Y , Oh-Tsu M , Sasaki M , Kimura G , Nomoto K
Published: January 2020

Cytomegalovirus (CMV) causes severe clinical manifestations in immunocompromised hosts; however, it remains unclear whether the virus itself is a cause of immunosuppression or whether it is involved as an opportunistic bystander pathogen. This study was performed to elucidate the effect of CMV infection on the host's immune system. The double-positive thymocytes of BALB/c mice inoculated with a sublethal dose of murine CMV (MCMV) were extensively depleted by a 10-micrograms amount of anti-CD3 monoclonal antibody, while such an amount was unable to induce any apparent elimination of thymocytes in noninfected mice. In immature thymocytes of infected hosts, a markedly high level of susceptibility to apoptosis induction was found on treatment with anti-CD3 monoclonal antibody. Analysis of the signal transduction pathway of such double-positive thymocytes demonstrated a profound elevation of the intracellular Ca2+ level after anti-CD3 stimulation, implying that this aberrant mobilization of Ca2+ plays a crucial role in the signaling pathway leading these cells to an extensive apoptosis. Examination of the thymus by PCR was able to detect a low copy number of MCMV DNAs in thymic stromal cells but none at all in thymocytes. Therefore, it is suggested that a mechanism which is not associated with virus replication within the cells exerts a critical effect on rendering the thymocytes highly apoptosis sensitive in hosts infected with MCMV.
Identification and characterization of the ICP22 protein of equine herpesvirus 1
Authors: Holden V R , Caughman G B , Zhao Y , Harty R N , O'Callaghan D J
Published: January 2020

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.
Identification and transcriptional regulation of the baculovirus lef-6 gene
Authors: Passarelli A L , Miller L K
Published: January 2020

We identified an Autographa californica nuclear polyhedrosis virus (AcMNPV) gene, the late expression factor 6 gene (lef-6), which is involved in expression from late and very late AcMNPV gene promoters but not from an early AcMNPV gene promoter in transient expression assays. This gene was located within the PstI I fragment of the AcMNPV genome (14.7 to 17.9 map units), immediately downstream of Ac-iap, the AcMNPV homolog of a baculovirus gene family involved in blocking apoptotic programmed cell death. The nature and temporal regulation of both Ac-iap and lef-6 transcripts was examined. Ac-iap and lef-6 were cotranscribed as bicistronic messages at both early and late times postinfection. In addition, lef-6 was transcribed as a monocistronic mRNA by initiation from an early promoter within Ac-iap.
Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses
Authors: Nédellec P , Dveksler G S , Daniels E , Turbide C , Chow B , Basile A A , Holmes K V , Beauchemin N
Published: January 2020

Murine coronaviruses such as mouse hepatitis virus (MHV) infect mouse cells via cellular receptors that are isoforms of biliary glycoprotein (Bgp) of the carcinoembryonic antigen gene family (G. S. Dveksler, C. W. Dieffenbach, C. B. Cardellichio, K. McCuaig, M. N. Pensiero, G.-S. Jiang, N. Beauchemin, and K. V. Holmes, J. Virol. 67:1-8, 1993). The Bgp isoforms are generated through alternative splicing of the mouse Bgp1 gene that has two allelic forms called MHVR (or mmCGM1), expressed in MHV-susceptible mouse strains, and mmCGM2, expressed in SJL/J mice, which are resistant to MHV. We here report the cloning and characterization of a new Bgp-related gene designated Bgp2. The Bgp2 cDNA allowed the prediction of a 271-amino-acid glycoprotein with two immunoglobulin domains, a transmembrane, and a putative cytoplasmic tail. There is considerable divergence in the amino acid sequences of the N-terminal domains of the proteins coded by the Bgp1 gene from that of the Bgp2-encoded protein. RNase protection assays and RNA PCR showed that Bgp2 was expressed in BALB/c kidney, colon, and brain tissue, in SJL/J colon and liver tissue, in BALB/c and CD1 spleen tissue, in C3H macrophages, and in mouse rectal carcinoma CMT-93 cells. When Bgp2-transfected hamster cells were challenged with MHV-A59, MHV-JHM, or MHV-3, the Bgp2-encoded protein served as a functional MHV receptor, although with a lower efficiency than that of the MHVR glycoprotein. The Bgp2-mediated virus infection could not be inhibited by monoclonal antibody CC1 that is specific for the N-terminal domain of MHVR. Although CMT-93 cells express both MHVR and Bgp2, infection with the three strains of MHV was blocked by pretreatment with monoclonal antibody CC1, suggesting that MHVR was the only functional receptor in these cells. Thus, a novel murine Bgp gene has been identified that can be coexpressed in inbred mice with the Bgp1 glycoproteins and that can serve as a receptor for MHV strains when expressed in transfected hamster cells.
Changes in the cytopathic effects of human immunodeficiency virus type 1 associated with a single amino acid alteration in the ectodomain of the gp41 transmembrane glycoprotein
Authors: Cao J , Vasir B , Sodroski J G
Published: January 2020

A human immunodeficiency virus type 1 mutant with a single amino acid change (designated 596 W/M) in the ectodomain of the gp41 transmembrane envelope glycoprotein replicated in T-cell lines and in CD4-positive peripheral blood mononuclear cells identically to the wild-type virus. However, the cytopathic effects associated with infection by the mutant virus were altered, with a marked attenuation of syncytium formation and a significant delay in single-cell lysis relative to those of the wild-type virus-infected culture. The 596 W/M mutant is apparently defective in a function that is dispensable for virus entry but that contributes to the efficiency of induction of viral cytopathic effects.
Heat shock response to vaccinia virus infection
Authors: Sedger L , Ruby J
Published: January 2020

We have investigated the induction of heat shock proteins (HSPs) in mice infected with vaccinia virus. Vaccinia virus replicates to high levels in the ovaries of infected mice and causes a significant inhibition of host cell DNA, RNA, and protein synthesis. Many HSPs are constitutively expressed in murine ovarian tissue at low levels, consistent with their obligatory role in normal physiological events. In contrast with these events, HSP expression was augmented in virus-infected mouse ovaries 6 days postinfection. In particular, there was a dramatic increase in the expression of a protein identified as the inducible 72-kDa HSP. Analysis of cellular mRNA confirmed this protein to be the major mouse inducible HSP70 and demonstrated its presence within virus-infected cells. Hence, we have demonstrated the expression of stress proteins during poxvirus infection in vivo.
Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P
Authors: Parks G D
Published: January 2020

The paramyxovirus large protein (L) and phosphoprotein (P) are both required for viral RNA-dependent RNA polymerase activity. Previous biochemical experiments have shown that L and P can form a complex when expressed from cDNA plasmids in vivo. In this report, L and P proteins of the paramyxovirus simian virus 5 (SV5) were coexpressed in HeLa T4 cells from cDNA plasmids, and L-P complexes were examined. To identify regions of the SV5 L protein that are required for L-P complex formation, 16 deletion mutants were constructed by mutagenesis of an SV5 L cDNA. Following coexpression of these L mutants with cDNA-derived P and radiolabeling with 35S-amino acids, cell lysates were analyzed for stable L-P complexes by a coimmunoprecipitation assay and by sedimentation on 5 to 20% glycerol gradients. Mutant forms of L containing deletions that removed as much as 1,008 residues from the C-terminal half of the full-length 2,255-residue L protein were detected in complexes with P by these two assays. In contrast, large deletions in the N-terminal half of L resulted in proteins that were defective in the formation of stable L-P complexes. Likewise, L mutants containing smaller deletions that individually removed N-terminal regions which are conserved among paramyxovirus and rhabdovirus L proteins (domain I, II, or III) were also defective in stable interactions with P. These results suggest that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions. SV5-infected HeLa T4 cells were pulse-labeled with 35S-amino acids, and cell extracts were examined by gradient sedimentation. Solubilized L protein was detected as an approximately 8 to 10S species, while the P protein was found as both a approximately 4S form (approximately 85%) and a species that cosedimented with L (approximately 15%). These data provide the first biochemical evidence in support of a simple domain structure for an L protein of the nonsegmented negative-sense RNA viruses. The results are discussed in terms of a structural model for the L protein and the interactions of L with the second viral polymerase subunit P.
Protein and DNA elements involved in transactivation of the promoter of the bovine herpesvirus (BHV) 1 IE-1 transcription unit by the BHV alpha gene trans-inducing factor
Authors: Misra V , Bratanich A C , Carpenter D , O'Hare P
Published: January 2020

In herpes simplex virus (HSV)-infected cells, the transcription of immediate-early (alpha) genes is regulated by a virion component, the alpha gene trans-inducing factor (alpha TIF). This protein forms a complex with cellular factors and TAATGARAT motifs present in one or more copies in the promoters of all alpha genes. We have characterized the bovine herpesvirus 1 (BHV-1) homolog of this protein. Like its HSV counterpart, the BHV alpha TIF was synthesized in the later stages of infection and could be demonstrated to be a component of purified virions. In transient expression assays, BHV alpha TIF was a strong transactivator and stimulated the activity of IE-1, the major BHV-1 alpha gene promoter, with an efficiency comparable to that of HSV alpha TIF. This stimulation was largely dependent on a TAATGAGCT sequence present in a single copy in IE-1, and BHV alpha TIF, in conjunction with cellular factors, formed a complex with oligonucleotides containing this sequence. Despite these similarities between the two alpha TIFs, our preliminary observations suggest that the proteins may activate transcription by different mechanisms. Although BHV alpha TIF strongly transactivated IE-1, it differed from its HSV counterpart in that the carboxyl terminus of BHV alpha TIF, when fused to the DNA-binding domain of GAL4, was a relatively poor stimulator of a promoter containing GAL4-binding sites. Also unlike HSV alpha TIF, removal of the carboxyl terminus of BHV alpha TIF reduced but did not eliminate the ability of the protein to transactivate IE-1. These results are discussed in view of the structural similarities and differences among the alpha TIFs of alphaherpes-viruses.
Nuclear localization of foamy virus Gag precursor protein
Authors: Schliephake A W , Rethwilm A
Published: January 2020

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus.
Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs
Authors: Tiley L S , Hagen M , Matthews J T , Krystal M
Published: January 2020

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.
Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10
Authors: Connors M , Giese N A , Kulkarni A B , Firestone C Y , Morse H C , Murphy B R
Published: January 2020

In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.