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V G Chinchar

Publications and source records attributed to V G Chinchar.

At least 37 records · Page 2Linked to original sources
PubMed1998

Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

Animals↗
PubMed1997

Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

Animals↗
PubMed1997

Molecular characterization, sequence analysis, and taxonomic position of newly isolated fish iridoviruses.

Within the past decade, iridoviruses have been identified as the causative agents of systemic disease in a variety of commercially and recreationally important fish. Here we examine nine iridoviruses from fish, reptiles, and amphibians and demonstrate that all isolates were more similar to frog virus 3, the type species of the genus Ranavirus, than to lymphocystis disease virus, the type species of the genus Lymphocystivirus. Comparison of viral protein synthesis profiles, restriction endonuclease digestion patterns, and the amino acid sequence of the major capsid protein indicated that iridoviruses isolated from the same geographic region were similar, if not identical, whereas viruses from different areas were distinct. Moreover, using primers complementary to the conserved major capsid protein, we found that both PCR and RT-PCR successfully amplified virus-specific nucleic acid from all nine isolates. These studies demonstrate that the piscine iridoviruses examined here were members of the genus Ranavirus, and suggest that surveys of pathogenic "fish viruses" may need to include neighboring amphibian and reptilian populations. In addition, the results indicate that PCR readily identified vertebrate iridoviruses and suggest that PCR will be useful in the diagnosis of fish disease.

Amino Acid Sequence↗
PubMed1996

Cloning, sequence analysis, and expression of the major capsid protein of the iridovirus frog virus 3.

The nucleotide sequence of the gene encoding in the major capsid protein (MCP) of frog virus 3 (FV3) has been determined and compared to other iridovirus capsid genes. Nucleotide sequence and S1 nuclease analysis showed that the FV3 MCP gene encoded a transcript of 1452 nucleotides containing a 12 nucleotide AU-rich 5' nontranslated region (NTR) and a 50-nucleotide 3' NTR whose terminus was predicted to fold into a hairpin of moderate stability. An open reading frame initiating from the the 5'-most AUG codon encoded a protein of 463 amino acids with a predicted molecular weight of 49,860. Expression of the putative FV3 MCP gene in vitro confirmed that it encoded the major capsid protein of FV3 and supported the suggestion that translation initiated at AUG-1. Pairwise amino acid alignments detected a high degree of sequence identity between the FV3 MCP and other iridoviruses. These results indicate that iridoviruses possess an evolutionarily related major capsid protein and provide information useful not only in studies of viral gene expression, but also in characterizing newly isolated iridoviruses.

Amino Acid Sequence↗
PubMed1996

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Animals↗
PubMed1994

Instability of frog virus 3 mRNA in productively infected cells.

Cloned DNA restriction fragments encoding representative frog virus 3 messages were used as probes to assess the stability of viral transcripts in infected fathead minnow cells. Analysis of Northern blot hybridization profiles confirmed earlier findings and revealed that in infected cells the steady-state level of representative frog virus 3 (FV3) messages increased throughout the replication cycle. However, when actinomycin D was added at 4 hr after infection to block the synthesis of new transcripts, viral messages were observed to turn over rapidly, with half-lives of approximately 2 hr. These results indicate that viral transcripts were not preferentially stabilized in FV3-infected cells and suggest that the high steady-state level of viral messages present at late times after infection was due to viral transcription outpacing message degradation. Moreover, the instability of viral messages challenges the suggestion that the terminal dyad symmetry (hairpin structure) observed in all frog virus 3 messages sequenced to date plays a role in transcript stability.

Animals↗
PubMed1993

Productive infection of continuous lines of channel catfish leukocytes by channel catfish virus.

Channel catfish virus (CCV) undergoes extensive replication in fingerling catfish and establishes latent infection in survivors. Although the site of viral latency in carriers is unknown, a variety of tissues, including leukocytes, have been implicated. To explore the interaction of CCV and leukocytes further, we examined in vitro infection of continuous lines of cloned catfish B cells and non-cloned lines containing predominantly macrophages and putative T-cells. Our results indicate that all three leukocyte subpopulations were productively infected with CCV, but that the kinetics of infection and the final yields differed markedly. These findings shed light on CCV-leukocyte interaction and suggest that CCV-infected autologous macrophage or T-cell lines might be suitable targets for assays of catfish cytotoxic T cell activity.

Animals↗
PubMed1992

Metabolism of host and viral mRNAs in frog virus 3-infected cells.

Treatment of purified frog virus 3 (FV3) with nonionic detergent and high salt released an endoribonucleolytic activity and confirmed earlier findings of a virion-associated endonuclease. This observation, coupled with evidence implicating host and viral message destabilization in herpesvirus and poxvirus biogenesis, raised the question of what role, if any, mRNA degradation plays in FV3 replication. To answer this question, Northern analyses of mock- and virus-infected cells were performed using probes for representative host and viral messages. These studies demonstrated that the steady state level of host messages progressively declined during the course of productive FV3 infection, whereas the steady state level of viral messages was not affected. To determine whether the decline in the steady state level of host mRNA was due to virus-induced degradation or to normal turnover coupled to virus-mediated transcriptional shut-off, actin mRNA levels were examined in mock- and virus-infected cells in the presence and absence of actinomycin D. Under these conditions, actin mRNA levels declined more quickly in actinomycin D-treated, virus-infected cells, than in mock-infected cells incubated in the presence of actinomycin D suggesting that the decline in the steady state level of actin mRNA was due to degradation. However, although it appears as if host message degradation is responsible for virus-mediated translational shut-off, the ability of heat-inactivated FV3 to block cellular translation without destabilizing cellular messages indicates that message degradation is not required for translational inhibition. As noted above, the degradation of early FV3 messages was not involved in controlling the transition from early to late gene expression. Furthermore, the presence of abundant, but nontranslated, early messages late in infection, coupled with the inefficient translation of late messages in vitro supported earlier suggestions that FV3 gene expression is controlled, at least in part, at the translational level. Taken together, these results suggest that FV3 regulates gene expression in a unique manner and may be a good model to examine the mechanics of translational control.

Animals↗
PubMed1991

Translational control of equine herpesvirus type 1 gene expression.

Translational control mechanisms modulate gene expression in a variety of cellular and viral systems. Using hypertonic conditions to block protein synthesis in vivo, we observed that the synthesis of several major equine herpesvirus type 1 proteins was selectively inhibited. Although sensitivity to hypertonic conditions was graded across a continuum, messages coding for proteins of 203, 130.5, and 31.5 kDa were significantly more resistant to higher salt concentrations in vivo than those coding for polypeptides of 148, 116, and 74 kDa. Similar results were observed in vitro when potassium acetate was used to block initiation. In addition, Northern blot analyses demonstrated that steady-state levels of cellular mRNAs declined beginning at about 6 hr after infection. Taken together, these results indicate that the expression of several major equine herpesvirus type 1 genes was controlled in part at the post-transcriptional level.

Acetates↗
PubMed1990

Translational efficiency: iridovirus early mRNAs outcompete tobacco mosaic virus message in vitro.

Infection with the iridovirus, frog virus 3, results in the rapid inhibition of host cell protein synthesis and is correlated with activation of an eIF-2 kinase. Because phosphorylation of eIF-2 inhibits ternary complex formation and thus reduces the overall level of translation, it has been suggested that frog virus 3 messages escaped translational shut-off by outcompeting host messages for the remaining translational capacity of the cell. In this report, we show that frog virus 3 messages were more translationally competitive than highly efficient tobacco mosaic virus transcripts based on their relative resistance to inhibitors of initiation. This result strengthens the suggestion that the selective translation of frog virus 3 transcripts in virus-infected cells may be a reflection of their enhanced competitiveness.

Animals↗
PubMed1990

Frog virus 3-mediated translational shut-off: frog virus 3 messages are translationally more efficient than host and heterologous viral messages under conditions of increased translational stress.

Frog virus 3 rapidly and selectively blocks host cell translation while synthesizing more than 60 virus-specific polypeptides. Previous work indicated that virus infection led to activation of a kinase that phosphorylated and, as a consequence, inactivated eIF-2. Although phosphorylation of eIF-2 could explain the rapid decline in host cell translation, it could not explain how viral protein synthesis persisted in the face of host shut-off. To explain this phenomenon, we speculated that viral messages, either as a consequence of their higher translational efficiency or their greater abundance, were able to outcompete host messages for the remaining translational initiation complexes. To test this hypothesis, the relative translational efficiency of three characteristic FV3 messages was measured against that of several model messages. Translational efficiency was determined by monitoring the resistance (and hence the competitiveness) of a given transcript to increasing concentrations of salt in vitro and in vivo. In both rabbit reticulocyte lysates and wheat germ extracts, FV3 messages were more resistant to supra-optimal concentrations of potassium acetate than globin message and three BMV transcripts. In vivo, FV3 polypeptides were synthesized in the presence of salt concentrations that blocked host cell protein synthesis. These results suggest that the selective translation of FV3 messages in virus-infected cells may partly be due to the higher translational efficiency of viral messages. Structural features that contribute to translational efficiency are discussed.

Acetates↗
PubMed1989

Frog virus 3-induced translational shut-off: activation of an eIF-2 kinase in virus-infected cells.

Infection of susceptible fathead minnow or Friend erythroleukemia cells with either infectious or heat-inactivated frog virus 3 led to the rapid inhibition of cellular protein synthesis. As seen in other cells, translational shut-off was accompanied by the dissociation of polysomes, but not the degradation of irreversible inactivation of cellular mRNAs. In addition, lysates from cells infected with heat-inactivated FV3 showed a reduced capacity to synthesize protein and to form 43S pre-initiation complexes in vitro. These results indicate that the in vitro systems accurately reflected in vivo events, and suggest that translational shut-off occurred prior to the union of the 40S ribosomal subunit and the [eIF-2.GTP.Met tRNAi] ternary complex. To determine the basis for the translational block, lysates from mock- and FV3-infected cells were assayed in vitro for their ability to phosphorylate the alpha subunit of eIF-2. In contrast to lysates from mock-infected cells, lysates from cells infected with heat-inactivated or infectious FV3 readily phosphorylated the alpha subunit of eIF-2. Since phosphorylation of the alpha subunit of eIF-2 inhibits its catalytic utilization during polypeptide chain initiation, these findings suggest that translational shut-off mediated by FV3 may be due to activation of a kinase that selectively phosphorylates this key initiation factor.

Animals↗
PubMed1989

Hemin and cyclic AMP stimulate message-dependent translation in lysates from Friend erythroleukemia cells.

A message-dependent, cell-free translation system was prepared from both uninduced and N,N'-hexamethylene-bis-acetamide-induced Friend erythroleukemia cells following modification of standard protocols. Active extracts were prepared by lysing Friend erythroleukemia cells in hypotonic buffer containing 50 microM hemin and 10 mM cyclic adenosine monophosphate (cAMP), and assaying translation in vitro in the absence of exogenous nucleoside triphosphates. Both hemin and cAMP were required for full activity and appeared to act by prolonging initiation in vitro. Following treatment of cell extracts with micrococcal nuclease, brome mosaic virus, globin, equine herpesvirus type 1, and frog virus 3 mRNA were efficiently translated to yield full-sized products. Using brome mosaic virus as a test message, levels of translation equivalent to 30%-50% of that seen in mock-treated lysates were obtained. These results attest to the translational efficiency of extracts prepared and assayed in the manner described above, and suggest that such extracts may be useful both for routine translational studies, and as a tool to dissect translational changes accompanying erythroid differentiation in vitro.

Acetamides↗
PubMed1986

Heat-inactivated frog virus 3 selectively inhibits equine herpesvirus type 1 translation in a temporal class-dependent manner.

Superinfection of equine herpesvirus type 1 (EHV-1)-infected rabbit kidney cells with heat-inactivated frog virus 3 (delta FV3) differentially blocked EHV-1 protein synthesis. The extent of inhibition varied with the specific EHV-1 message, but in general late protein synthesis was inhibited more than early and immediate early translation. Since FV3 has been shown to block heterologous RNA and protein synthesis, it was necessary to determine whether the observed reduction in herpesvirus protein synthesis was primarily due to a block in translation or to an earlier inhibition of EHV-1 mRNA synthesis. To distinguish between these alternatives, replicate cultures of EHV-1 infected cells were either superinfected with delta FV3 or treated with 10 micrograms/ml actinomycin D at 6 hr after infection, and EHV-1 protein synthesis monitored 3 hr later. We found that addition of actinomycin D to EHV-1 infected cultures had only a slight effect on EHV-1 translation, whereas superinfection with delta FV3 markedly reduced EHV-1 protein synthesis. This result suggested that the observed decline in EHV-1 protein synthesis was not due to the inhibition of herpesvirus mRNA synthesis. In addition, we showed that RNA extracted from delta FV3-superinfected cells directed the synthesis of full-size EHV-1 proteins in vitro indicating that shut-off was not caused by the degradation of EHV-1 mRNAs. Taken together these results show that delta FV3 selectively inhibited EHV-1 protein synthesis and are consistent with earlier observations which suggest that translational shut-off occurs at initiation.

Animals↗
PubMed1986

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization.

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units.

Antigens, Viral↗
PubMed1984

Isolation and characterization of a frog virus 3 variant resistant to phosphonoacetate: genetic evidence for a virus-specific DNA polymerase.

A variant of frog virus 3 (FV3) resistant to 200 micrograms/ml phosphonoacetate was isolated, and used to establish that the DNA polymerase induced in FV3-infected cells was virus coded. In addition, inhibitor studies showed that the FV3 polymerase is similar to eukaryotic polymerase alpha in its sensitivity to aphidicolin, and that resistance to phosphonoacetate does not confer cross-resistance to thymidine arabinoside or acycloguanosine.

Animals↗