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PubMed1996

Partial inhibition of yellow fever virus replication in vitro with different phosphorothioate oligodeoxyribonucleotides.

Phosphorothioate oligodeoxyribonucleotides targeted to different regions of the viral genome were synthesized and used in two kinds of experiments testing their activity against yellow fever virus (YFV) replication in cultured cells. We found that oligonucleotides complementary to the 3'-end or to the coding region of the viral RNA were regularly active in plaque reduction assay, although with inconstant efficiency. Oligonucleotides targeted to the 5'-end or to the initiation codon region exhibited lesser activity. Homologous oligonucleotides targeted to dengue virus RNA had no detectable inhibitory activity against dengue virus replication. However, in YFV production reduction assay, a non-specific inhibitory activity of a random oligonucleotide was observed. Taken as a whole, our results indicate that flaviviruses present detectable but heterogeneous sensitivity to phosphorothioate inhibition. Possible explanations are discussed.

Animals↗

PubMed1997

Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking.

The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. Deletion of both gI and gE prevented virus replication. Complete deletion of gI or deletions of 60% of the N terminus or 40% of the C terminus of gI resulted in a small plaque phenotype as well as reduced yields of infectious virus. Melanoma cells infected with gI deletion mutants formed abnormal polykaryocytes with a disrupted organization of nuclei. In the absence of intact gI, gE became localized in patches on the cell membrane, as demonstrated by confocal microscopy. A truncated N-terminal form of gI was transported to the cell surface, but its expression did not restore plaque morphology or infectivity. The fusogenic function of gH did not compensate for gI deletion or the associated disruption of the gE-gI complex. These experiments demonstrated that gI was dispensable for VZV replication in vitro, whereas gE appeared to be required. Although VZV gI was dispensable, its deletion or mutation resulted in a significant decrease in infectious virus yields, disrupted syncytium formation, and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a nonnative locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient membrane fusion during VZV replication.

Biological Transport↗

PubMed1996

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.

Amino Acid Sequence↗

PubMed2006

Chronic morphine exposure causes pronounced virus replication in cerebral compartment and accelerated onset of AIDS in SIV/SHIV-infected Indian rhesus macaques.

Six morphine-exposed and 3 control male Indian rhesus macaques were intravenously inoculated with mixture of SHIV(KU), SHIV(89.6)P and SIV/17E-Fr. These animals were followed for a period of 56 weeks in order to determine CD4 and CD8 profile, viral loads in plasma and cerebrospinal fluid (CSF), relative distribution of 3 pathogenic viruses in blood and brain, binding as well neutralizing antibody levels and cellular immune responses. Both morphine-exposed and control macaques showed a precipitous loss of CD4+ T cells; control animals, however, showed a greater tendency to recover these cells than did their morphine-exposed counterparts. The plasma and CSF viral loads were significantly higher in morphine-exposed group than those in the control group. Four morphine-exposed animals succumbed to SIV/SHIV-induced AIDS at week 18, 19, 20 and 51; post-infection with neurological disorders was found in 3 of the 4 animals. At the end of the 56-week observation period, 2 morphine-exposed and 3 control animals were still alive. All 3 viruses replicated in the blood of both morphine-exposed and control macaques, but the cerebral compartment showed a selection phenomenon; only SIV/17E-Fr and SHIV(KU) successfully crossed the blood brain barrier (BBB). The morphine-exposed macaques further favored viral migration through the blood brain barrier (BBB). SIV/17E-Fr crossed the BBB within 2 weeks in both morphine-exposed and control macaques, whereas SHIV(KU) crossed the BBB more rapidly in morphine-exposed than in control macaques. Three morphine-exposed macaques (euthanized at weeks 18, 19 and 20) did not develop cellular or humoral immune responses, whereas the other 3 morphine-exposed and 3 control macaques developed both cellular and humoral immune responses.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Studies on mechanism of action of glycyrrhizin against hepatitis A virus replication in vitro.

Glycyrrhizin (GL) achieved a concentration-dependent inhibition of the replication of hepatitis A virus (HAV) in PLC/PRF/5 cells. GL has been shown to inhibit an early stage of the HAV replication. GL was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labelled virions to cells. GL inhibited HAV penetration of the plasma membrane as measured by the amount of infective virus no longer neutralizable by specific antibody over time.

Antigens, Viral↗

PubMed2000

Significant interference with hepatitis B virus replication by a core-nuclease fusion protein.

Hepatitis B virus (HBV), a small DNA containing virus that replicates via reverse transcription, causes acute and chronic B-type hepatitis in humans. The limited success of current therapies for chronic infection has prompted exploration of alternative strategies. Capsid-targeted viral inactivation is a conceptually powerful approach that exploits virion structural proteins to target a degradative enzyme specifically into viral particles. Its principal feasibility has been demonstrated in retroviral model systems but not yet for a medically relevant virus outside the retrovirus family. Recently, we found that C proximal fusion to the HBV capsid protein of the Ca(2+)-dependent nuclease (SN) from Staphylococcus aureus yields a chimeric protein, coreSN, that in Escherichia coli coassembles with the wild-type capsid protein into particles with internal SN domains. Here we show that, in HBV co-transfected human hepatoma cells, less than 1 coreSN protein per 10 wild-type core protein subunits reduced titers of enveloped DNA containing virions by more than 95%. The antiviral effect depends on both an enzymatically active SN and on the core domain. CoreSN does not block assembly of RNA containing nucleocapsids but interferes with proper synthesis of viral DNA inside the capsid, or leads to rapid DNA degradation. Our data suggest an intracellular nuclease activation that, owing to the characteristics of HBV morphogenesis, is nonetheless highly virus specific. HBV may therefore be particularly vulnerable to the capsid-targeted viral inactivation approach.

Antiviral Agents↗

PubMed1988

Acute and chronic hepatitis delta virus infection: direct or indirect effect on hepatitis B virus replication?

In a large population of patients, chronic hepatitis delta virus (HDV) infection was usually associated with absence of hepatitis B virus (HBV) replication. However, acute HDV superinfection progressing to chronic HDV infection in two hepatitis B e antigen (HBeAg)-positive HBV carriers and coinfection in two other patients who progressed to chronic HBV (HBeAg-positive) and HDV infection was associated with continuing high-level HBV replication for several years. Thus HDV infection does not always inhibit HBV replication. The hypothesis that the different effects of HDV coinfection and superinfection on HBV replication may stem from variability in the capacity of the host to produce and respond to interferon is discussed.

Acute Disease↗

PubMed2003

Intermediate filament integrity is required for Junin virus replication.

The role of the cytoskeletal framework in Junin virus (JUNV) replication has already been demonstrated with compounds interfering with the microfilament (MF) and microtubule (MT) networks. In this work, we evaluated the role of intermediate filaments (IF) during JUNV infection. We tested the effect of acrylamide, a compound that selectively disrupts IF, in culture of three different cell types: Vero cells, murine astrocytes and human foreskin fibroblasts. Perturbation of intermediate filaments had an inhibitory effect on JUNV production within a range of acrylamide concentration of 0.5-3mM in a dose-dependent manner, without cell viability modification. Recovery experiments showed that viral production was partially increased when medium containing acrylamide was replaced by normal maintenance medium (MM). The adsorption and internalization steps were not affected by IF disruption. The expression of JUNV proteins was highly reduced in the presence of 2mM acrylamide while immunofluorescence staining of IF showed network disruption with the formation of cytoplasmic aggregates containing vimentin or glial fibrillary acidic protein (GFAP). We conclude that the IF network may play a role in the early step of JUNV multiplication, subsequent to virus entry and that its integrity is a necessary condition for the normal replication of JUNV in neural and fibroblast cells as well as in the Vero cell line.

Acrylamide↗

PubMed2004

CD38 expression on CD8 T lymphocytes as a marker of residual virus replication in chronically HIV-infected patients receiving antiretroviral therapy.

The level of CD8+ CD38+ T lymphocytes in blood correlates with disease progression in HIV-infected individuals, independently of the CD4 count. Effective antiretroviral therapy reduces this lymphocyte subset in parallel with plasma viremia, although CD38 expression on CD8+ cells does not normalize completely in most subjects, and might reflect residual HIV replication. The expression of CD38 on CD8+ cells (as number of CD38 molecules per CD8+ cell) was measured quantitatively by flow cytometry in 200 individuals, of whom 170 were HIV positive and 30 were HIV-uninfected controls. Forty-six HIV-infected subjects were on antiretroviral therapy and had undetectable viral load. The remaining 124 HIV-positive persons were not on therapy and had detectable plasma viremia. The mean level of CD38 on CD8+ cells was higher in HIV-positive, untreated patients than in subjects on antiviral therapy and controls (5023, 2029, and 1978 molecules per CD8+ cell, respectively, p < 0.01). In HIV-positive, untreated subjects, the higher CD38 expression mainly occurred on CD45RO+ CD8+ cells. The level of CD38 strongly correlated with plasma HIV-RNA (r = 0.63, p < 0.001). The levels of CD38 on CD8+ cells declined steadily in HIV-positive subjects after beginning antiretroviral therapy. A few individuals presented viral blips whereas being on antiviral treatment, levels of CD38 on CD8+ cells increased transiently in parallel with episodes of viral replication. Levels of CD38 on CD8+ cells are increased in chronic HIV infection, and strongly correlate with plasma viremia. The slow decline of CD38 expression on CD8+ cells over time in subjects with undetectable plasma viremia while being on antiretroviral therapy suggests that CD38 expression on CD8+ cells could be used as a marker of residual virus replication.

ADP-ribosyl Cyclase↗

PubMed1971

Mumps virus replication in chick embryo lung cells: properties of ribonucleic acids in virions and infected cells.

Ribonucleic acid (RNA) species in mumps virions and in infected cells were compared. The predominant RNA species in virions labeled with (3)H-uridine sedimented at 50S; RNA species sedimenting at 28, 18, and about 10S were also present. The virion-associated RNA species sedimenting slower than 50S contained some nucleotide sequences similar to 50S virion RNA. Although mumps virus replication was severely inhibited by high concentrations of actinomycin D, some virus was made, and virus-specific RNA species accumulated in infected cells. Mumps virus resembled other paramyxoviruses in inducing, in infected cells, synthesis not only of 50S RNA but also of slower sedimenting RNA species with a peak distribution at about 18S, complementary in base sequences to 50S virion RNA. In addition, base sequences of the parental type were relatively abundant in the RNA species sedimenting slower than 50S; these may represent precursors of the slowly sedimenting RNA species associated with virions. Ribonuclease-resistant RNA was detected in infected cells; this may represent replicative or transcriptive intermediates. Inhibition of protein synthesis with cycloheximide severely depressed accumulation of labeled 50S RNA in infected cells but did not interfere with accumulation of RNA species sedimenting slower than 50S. Actinomycin D treatment had a similar effect. Annealing of genomes and virus-induced complementary RNA species of Newcastle disease virus, Sendai virus, and mumps virus did not reveal any base sequence homologies.

Animals↗

PubMed2005

Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

Avian metapneumovirus (aMPV) is one of the major causes of serious respiratory infections of poultry and leads to considerable economic losses to food animal production worldwide. Here, we show that double stranded short interfering RNA (siRNA) molecules corresponding to aMPV phosphoprotein (P) gene silence P RNA and protein expression. These siRNAs broadly reduced the expression of other viral proteins in addition to P, but did not have a discernable effect on cellular protein expression. The exposure of cells to P-specific siRNAs also led to inhibition of virus replication as evidenced by marked reduction in the progeny virion titers. Taken together, the findings suggest that exogenous P silencing siRNAs can inhibit aMPV replication with potential implications in the design of novel siRNA based prophylactics.

Animals↗

PubMed2003

Vaccinia virus uracil DNA glycosylase has an essential role in DNA synthesis that is independent of its glycosylase activity: catalytic site mutations reduce virulence but not virus replication in cultured cells.

Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.

Amino Acid Sequence↗

PubMed1999

Specific inhibition of hepatitis B virus replication by sense RNA.

We describe effects of sense RNA molecules on hepatitis B virus (HBV) replication and antigen synthesis in transiently transfected cells. When certain subgenomic fragments of HBV were expressed as sense RNA together with a replication-competent genome of HBV, they inhibited HBV replication by up to 75% and HBsAg secretion by up to 60%. The corresponding antisense sequences had a 50% inhibitory effect in one case and no effect in another case. The sense RNA species did not inhibit duck hepatitis B virus (DHBV) replication, suggesting specific inhibitory effects. HBV transcript levels were unaltered in the presence of sense RNA species, consistent with an inhibitory effect mediated at the posttranscriptional level. The inhibition of HBV replication by overexpression of sense RNA derived from the viral genome represents an example of sense cosuppression of an animal virus.

Cell Line↗

PubMed1987

Genetically determined resistance to lethal murine cytomegalovirus infection is mediated by interferon-dependent and -independent restriction of virus replication.

Susceptibility of 4-week-old mice of different strains to lethal murine cytomegalovirus (MCMV) infection was studied. Strains homozygous for H-2k and C57BL strains were resistant to greater than or equal to 10(5.5) PFU. B10.BR mice congenic for C57BL background genes and H-2k were about 10-fold more resistant than either C3H/HeN or C57BL strains. BALB/c mice (H-2d) were susceptible (50% lethal dose, 10(5.05) PFU). This susceptibility was dominant over resistance associated with H-2k but not that associated with C57BL background genes. The dominant susceptibility trait segregated in backcross mice as if carried by a single gene. Virus replication in spleen cells in vivo correlated with susceptibility to lethal infection. A similar trend was found in tests of salivary glands. Replication of MCMV in vitro in cultures of adherent spleen cells and primary mouse embryo cells correlated with replication in vivo. Neutralization of interferon (IFN) in cultures of adherent spleen cells reversed H-2k-linked restriction of viral replication but had minor effects on cells of other strains. Natural killer cell responses to infection were often higher in more resistant strains, but B10.BR mice developed minimal natural killer cell responses. Specific antibody and cytotoxic T cell responses in B10.BR mice were similar or lower than in other strains. Thus, resistance to lethal MCMV infection was not immunologically mediated, was dependent on and reflected by the capacity of cells from a given mouse strain to support replication in vivo and in vitro, and was IFN dependent and recessive if linked to H-2k but IFN independent when associated with C57BL background genes.

Animals↗

PubMed2005

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture.

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

Antiviral Agents↗

PubMed2001

Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.

Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

Cell Fusion↗

PubMed2005

Covert human immunodeficiency virus replication in dendritic cells and in DC-SIGN-expressing cells promotes long-term transmission to lymphocytes.

HIV-1 virions are efficiently captured by monocyte-derived immature dendritic cells (iDCs), as well as by cell lines expressing the lectin DC-SIGN. Viral infectivity can be retained for several days, and even enhanced, before transmission to CD4+ lymphocytes. The role of DC-SIGN in viral retention and enhancement of infection is not fully understood and varies according to the cell line expressing the lectin. We studied here the mechanisms underlying this process. We focused our study on X4-tropic human immunodeficiency virus (HIV) strains, since they were widely believed not to replicate in iDCs. However, we first show that X4 HIV replicates covertly and slowly in iDCs. This is also the case in Raji-DC-SIGN cells, which are classically used to study HIV transmission. We used either single-cycle or replicative HIV and measured viral RT and replication to further demonstrate that transfer of incoming virions from iDCs or DC-SIGN+ cells occurs only on the short-term (i.e., a few hours after viral exposure). There is no long-term storage of original HIV particles in these cells. A few days after viral exposure, replicative viruses, and not single-cycle virions, are transmitted to CD4+ cells. The cell-type-dependent activity of DC-SIGN reflects the ability of HIV to replicate covertly in some cells, and not in others.

B-Lymphocytes↗

PubMed2006

AcMNPV ORF38 protein has the activity of ADP-ribose pyrophosphatase and is important for virus replication.

The ORF38 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), or AcORF38, contains a conserved motif of Nudix (nucleotide diphosphate X) superfamily. It has the highest homology with ADP-ribose pyrophosphatase (ADPRase), a subfamily of Nudix pyrophosphatase. In the current study, recombinant AcORF38 protein was prepared and shown to have ADPRase activity, with a Km of 204 microM, and K(cat) of 6.96 s(-1) at pH 8.0 and 5 mM MgCl2. The transcription of AcORF38 was detected 2 h postinfection, and lasted until the late stage. An orf38 gene-deleted mutant virus, vAcGFP-Delta38, was constructed. Although it produced progeny virus, the yield of extracellular virus was less than 1% of the wild-type virus. The activity of viral very late gene promoter was also greatly reduced in vAcGFP-Delta38-infected cells as indicated by the expression of green fluorescence protein gene driven by polyhedrin promoter. The mutant phenotype was rescued by co-transfection with an AcORF38-expressing plasmid. These results suggest that AcORF38 plays an important role in virus replication, although the detail is to be elucidated.

Amino Acid Sequence↗