Agents useful in the study of mechanisms of viral replication. Discussion.
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Amantadine↗
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Results for “viral replication”
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Retroviral recombination in vivo: viral replication patterns and genetic structure of simian immunodeficiency virus (SIV) populations in rhesus macaques after simultaneous or sequential intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef.
To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Deltavpx/Deltavpr and then with SIVmac239Deltanef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Deltanef when plasma SIVmac239Deltavpx/Deltavpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Deltanef, and then with SIVmac239Deltavpx/Deltavpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.
Animals↗
Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.
The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.
3C Viral Proteases↗
Posttranscriptional effect of dexamethasone and interferon on the viral replication induced by iododeoxyuridine from murine transformed by nonproducer fibroblasts.
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Animals↗
Pattern of disease after murine hepatitis virus strain 3 infection correlates with macrophage activation and not viral replication.
Murine hepatitis virus strain (MHV-3) produces a strain-dependent pattern of disease which has been used as a model for fulminant viral hepatitis. This study was undertaken to examine whether there was a correlation between macrophage activation and susceptibility or resistance to MHV-3 infection. Peritoneal macrophages were isolated from resistant A/J and susceptible BALB/cJ mice and, following stimulation with MHV-3 or lipopolysaccharide (LPS), analyzed for transcription of mRNA and production of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), mouse fibrinogen-like protein (musfiblp), tissue factor (TF), leukotriene B4, and prostaglandin E2 (PGE2). Macrophages from BALB/cJ mice produced greater amounts of IL-1, TNF-alpha, TGF-beta, leukotriene B4, and musfiblp following MHV-3 infection than macrophages from resistant A/J mice, whereas in response to LPS, equivalent amounts of IL-1, TNF-alpha, TGF-beta, and TF were produced by macrophages from both strains of mice. Levels of mRNA of IL-1, TNF-alpha, and musfiblp were greater and more persistent in BALB/cJ than in A/J macrophages, whereas the levels and kinetics of IL-1, TNF-alpha, and TF mRNA following LPS stimulation were identical in macrophages from both strains of mice. Levels of production of PGE2 by MHV-3-stimulated macrophages from resistant and susceptible mice were equivalent; however, the time course for induction of PGE2, differed, but the total quantity of PGE2 produced was insufficient to inhibit induction of musfiblp, a procoagulant known to correlate with development of fulminant hepatic necrosis in susceptible mice. These results demonstrate marked differences in production of inflammatory mediators to MHV-3 infection in macrophages from resistant A/J and susceptible BALB/cJ mice, which may explain the marked hepatic necrosis and fibrin deposition and account for the lethality of MHV-3 in susceptible mice.
Animals↗
Ongoing viral replication is required for gammaherpesvirus 68-induced vascular damage.
The role of autoimmunity in large-vessel vasculitis in humans remains unclear. We have previously shown that infection of gamma interferon receptor knockout (IFN-gamma R(-/-)) mice with gammaherpesvirus 68 (gamma HV68) results in severe inflammation of the large elastic arteries that is pathologically similar to the lesions observed in Takayasu's arteritis, the nongranulomatous variant of temporal arteritis, and Kawasaki's disease (K. E. Weck et al., Nat. Med. 3:1346-1353, 1997). Here we define the mechanism of damage to the elastic arteries. We show that there is a persistent productive infection of the media of the large elastic vessels. In addition, we demonstrate that persistent virus replication is necessary for chronic arteritis, since antiviral therapy of mice with established disease resulted in increased survival, clearance of viral antigen from the media of the affected vessel, and dramatic amelioration of arteritic lesions. These data argue that ongoing virus replication, rather than autoimmunity, is the cause of gamma HV68-induced elastic arteritis.
Animals↗
The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication.
The herpes simplex virus DNA polymerase is composed of two subunits, a large catalytic subunit (Pol) and a smaller subunit (UL42) that increases the processivity of the holoenzyme. The interaction between the two polypeptides is of interest both for the mechanism by which it enables the enzyme to synthesize long stretches of DNA processively and as a possible target for the rational design of novel antiviral drugs. Here, we demonstrate through a combination of insertion and deletion mutagenesis that the carboxy-terminal 35 amino acids of Pol are crucial for binding UL42. The functional importance of the interaction was confirmed by the finding that a pol mutant defective for UL42 binding retained polymerase activity, but did not synthesize longer DNA products in the presence of UL42. Moreover, several association-incompetent mutants failed to complement the replication of a pol null mutant in a transient transfection assay, confirming that the Pol-UL42 interaction is necessary for virus replication in vivo and therefore a valid target for directed drug design.
Amino Acid Sequence↗
Differential effects of the splice acceptor at nucleotide 3295 of human papillomavirus type 31 on stable and transient viral replication.
In human papillomavirus type 31 (HPV-31), the E1--E4 and E5 open reading frames are expressed from polycistronic mRNAs. The major polycistronic mRNAs which encode E1--E4 and E5 are spliced messages which utilize a splice acceptor at nucleotide (nt) 3295 (SPA3295). Our laboratory recently developed a recombinant system for the synthesis of HPVs following immortalization of primary keratinocytes with cloned HPV-31 genomes (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). These immortalized cell lines are capable of maintaining HPV-31 DNA as episomes and induce the synthesis of virions in organotypic raft culture. In this study, we used these methods to begin an analysis of the roles of E1--E4 and E5 in HPV pathogenesis by mutating the major splice at nt 3295. Mutation of SPA3295 did not significantly alter the ability of HPV-31 genomes to replicate transiently in keratinocytes, nor did the mutation affect the immortalization potential of HPV-31. However, genomes carrying the SPA3295 mutation were not stably maintained as viral episomes, and the resulting immortalized keratinocyte cell line contained multiple, integrated copies of the mutated HPV-31 DNA. Northern analysis indicated that cell lines immortalized with the mutant HPV-31 expressed transcripts which were similar in size and abundance to wild-type messages, including those transcripts which rely on utilization of SPA3295. RNase protection and reverse transcription-PCR revealed that mutation of SPA3295 resulted in the utilization of a cryptic splice acceptor at nt 3298. These data suggest that the requirements for stable maintenance of HPV genomes are more stringent than those for transient replication and that factors which define these requirement rely on the major splice acceptor at nt 3295.
Alternative Splicing↗
Effects of gangliosides on the growth of herpes simplex virus type 1-infected cells derived from neurons and on viral replication.
Although it is well-known that herpes simplex virus can establish latent infections in neurons of sensory and sympathetic ganglia, little is known about the viral or cellular factors which regulate the latent state. Experiments were designed to elucidate the effects that can be produced by adding gangliosides, abundant components in neurons but not in most other cell types, to virus-infected cells from mouse trigeminal ganglia and from the neuroblastoma x glioma hybrid cell line NG108-15. The results obtained indicate that gangliosides, when used in combination with acyclovir, efficiently protect the infected cells from lysis in both cell systems, and that they can exert antiviral activity at least in part via suppressing protein kinase C activity.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Reciprocal enhancement of gene expression and viral replication between human cytomegalovirus and human immunodeficiency virus type 1.
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Bone Neoplasms↗
Immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus.
Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627-9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840-3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10(3)-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.
Animals↗
Saccharomyces cerevisiae: a useful model host to study fundamental biology of viral replication.
Understanding the fundamental steps of virus life cycles including virus-host interactions is essential for the design of effective antiviral strategies. Such understanding has been deferred by the complexity of higher eukaryotic host organisms. To circumvent experimental difficulties associated with this, systems were developed to replicate viruses in the yeast Saccharomyces cerevisiae. The systems include viruses with RNA and DNA genomes that infect plants, animals and humans. By using the powerful methodologies available for yeast genetic analysis, fundamental processes occurring during virus replication have been brought to light. Here, we review the different viruses able to direct replication and gene expression in yeast and discuss their main contributions in the understanding of virus biology.
Animals↗
The role of avian retroviral LTRs in the regulation of gene expression and viral replication.
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Alpharetrovirus↗
Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection.
The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = 0.0017), whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.
Antibodies, Monoclonal↗
Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.
The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.
Amino Acid Sequence↗
Response of sublethally irradiated monkeys to a replicating viral antigen.
Temporal effects of exposure to sublethal, total-body X radiation (400 R) on responses to vaccination with attenuated Venezuelan equine encephalomyelitis vaccine virus. TC-83, were examined in rhesus monkeys. Viremia, often with delayed onset, was prolonged even when irradiation preceded vaccination by 28 days. Virus titers were increased, articularly in groups irradiated 4 or 7 days before vaccination. Delay in appearance of hemagglutination-inhibition and serum-neutralizing antibody correlated closely with persistence of viremia in irradiated-vaccinated monkeys. The temporal course of antibody response was markedly affected by the intervals between irradiation and injection of this replicating antigen. With longer intervals between irradiation and vaccination, the somewhat depressed antibody responses approached normal or surpassed those of nonirradiated monkeys. Vaccination 14 days after radiation exposure resulted in lethality to 8 of 12 monkeys, apparently as a result of secondary infection. The additional lymphopenic stress due to the effect of TC-83, superimposed on the severly depressed hematopoietic competence at 14 days, undoubtedly contributed to this increased susceptibility to latent infection.
Animals↗
Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway.
Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.
Cell Nucleus↗
Viral replication in HeLa/fibroblast hybrid cells infected with human cytomegalovirus.
Three human hybrid cell lines were generated by the fusion of D 98OR, a HeLa cell variant, and TIG human diploid fibroblasts. Chromosome numbers of the hybrid cells fell between that of D 98OR cells and the combined chromosome number of the two cell lines, with three marker chromosomes identical to those of D 98OR cells. One hybrid, B-3, produced large amounts of human fibronectin as analyzed by immunohistochemistry. Only human cytomegalovirus (HCMV) infected B-3 cells showed positive fluorescence as detected by human antiserum to HCMV. Further cloning of B-3 by limiting dilution resulted in two cloned hybrids with markedly enhanced virus production as compared with B-3 cells. Treatment of these two clones with phorbol ester further enhanced virus production. These cloned hybrids may provide a tool to analyze host cell factors controlling the transcription and replication of HCMV.
Antigens, Viral↗