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PubMed1998

Nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line.

To better understand the early biochemical events that occur in human rhinovirus (HRV) infections, we examined the kinetics and mechanisms of interleukin-8 (IL-8) and IL-6 production from infected epithelial cells. Several HRV strains caused IL-8 and IL-6 production, but HRV-16 induced maximal IL-8 and IL-6 mRNA expression and protein production more rapidly than did HRV-14, despite similar rates of replication of the two viral strains. Viral induction of cytokine mRNA does not require new protein synthesis, since it was unaffected by cycloheximide treatment. The potent glucocorticoid budesonide did not affect viral replication or cytokine mRNA induction but modestly inhibited cytokine protein production. Interestingly, the nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (NONOate) inhibited both rhinovirus replication and cytokine production in a dose-dependent fashion without reducing levels of cytokine mRNA. The NONOate effects were due to release of nitric oxide, because NONOate that had been depleted of its nitric oxide content had no effect. Thus, nitric oxide may play an important anti-inflammatory and antiviral role in colds and nitric oxide donors may represent a novel therapeutic approach.

Cell Line↗
PubMed1987

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.

Base Sequence↗
PubMed2005

Evaluation of viral replication in children with chronic hepatitis B with and without interferon treatment.

BACKGROUND: In chronic infection with hepatitis virus B the fact that HBeAg becomes negative does not always mean suppression of viral replication. METHOD: HBV replication was assessed in 74 patients with chronic hepatitis or viral B cirrhosis, in whom diagnosis was made according to clinical, biological, and histological criteria. The patients were divided into two groups: group I (36 patients with interferon- therapy, 3 million U/m 2/ dose, 3 doses/week over a period of 4-6 months) and group II (control group of 38 patients who did not undergo interferon therapy). After a follow up period of 6 years in which patients underwent clinical, biochemical and serologic monitorization, HBV DNA was detected by the hybridization method on solid medium. RESULTS: During evolution the levels of transaminases became normal in both groups. The HBe Ag/Ab seroconversion rate at the end of the interferon therapy was 52.8% and the spontaneous HBe Ag/Ab seroconversion rate was 72.7% in group II after an average evolution of 6 years. HBs Ag/Ab seroconversion was not detected in any patient. Assessment of viral replication by HBV DNA testing at the end of the follow up period showed higher levels as compared to the HBeAg testing (69.4% vs. 25% in group I, 55.2% vs. 7.9% in group II). The absence of viral replication (HBV DNA negative) had similar rates in both groups (30.6% in group I vs. 44.8% in group II, p>0.9) and HBV DNA titers in the two groups were not significantly different at the end of the follow up period. In both groups, HBV DNA titers were significantly higher in patients with positive HBeAg. The concordance between the two viral markers was 100%. CONCLUSION: Because of the fluctuating evolution, long-term follow up and monitorization (including HBV DNA testing) of patients with chronic hepatitis B and of inactive HBsAg carriers are necessary.

Antiviral Agents↗
PubMed2005

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication.

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.

Animals↗
PubMed2005

Murine gammaherpesvirus 68 open reading frame 45 plays an essential role during the immediate-early phase of viral replication.

Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.

Amino Acid Sequence↗
PubMed1999

The primate lentivirus-encoded Nef protein can regulate several steps of the viral replication cycle.

The primate lentiviruses encode a protein, Nef, which is required for efficient viral replication in their host. Several biological activities of nef identified in vitro may contribute to this requirement in vivo, including receptor modulation and interference with cellular signaling pathways. We show that HIV- and SIV-encoded Nef can enhance virus production within a single viral replication cycle, not only by increasing viral infectivity, as previously reported, but also by acting through the efficiency of viral transcription and of viral release.

Animals↗
PubMed1991

[Study of viral replication in HbsAg positive chronic hepatitis].

Viral replication seric markers (VRSM), (BeHAg, BcHAg, and DNA-VHB) and the hepatic tissue BcHAg of thirty four patients suffering from chronic hepatopathies (6 PCH, 18 ACH, and 10 cirrhosis) were investigated. The greatest incidence of RVSM was observed in PCH. The combined study using all the markers demonstrated a positive correlation: DNA-VHB and BeHAg (p less than 0,001); DNA-VHB and BcHAg (p less than 0,001); BeHAg and BcHAg (p less than 0,001). The same result was obtained when comparing BcHAg (n) with the rest of RVSM: DNA-VHB (p less than 0,001), BeHAg (p less than 0,001) and seric BcHAg (p less than 0,01). Patients with a most intense histologic activity according to the Knodell index, presented a higher ratio of positive nuclei for BcHAg. The conclusion is that there is a good correlation amongst replication markers, specially hepatic BcHAg and DNA-VHB.

Biomarkers↗
PubMed1994

Characterization of hepatitis B virus core mutants that inhibit viral replication.

We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.

DNA Replication↗
PubMed2004

Oncolysis by viral replication and inhibition of angiogenesis by a replication-conditional herpes simplex virus that expresses mouse endostatin.

BACKGROUND: In preclinical models, infection of tumors by oncolytic strains of herpes simplex virus 1 (HSV-1) resulted in the destruction of tumor cells by viral replication and release of progeny virion that infected and destroyed adjacent tumor cells. However, complete tumor regression was rarely observed. METHODS: To augment the antitumor effect of viral oncolysis, a replication conditional HSV-1 mutant (HSV-Endo) was constructed in which the murine endostatin gene was incorporated into the HSV-1 genome. RESULTS: Replication of HSV-Endo effectively destroyed several colon carcinoma cell lines in vitro. Secretion of endostatin by HSV-Endo-infected HT29 human colon carcinoma cells was confirmed by Western blot analysis. The secreted endostatin was biologically active as assessed in a chick chorioallantoic membrane assay. Importantly, endostatin production at the site of viral replication did not inhibit viral replication. Direct injection of HSV-Endo into flank tumors caused tumor destruction, and some of the HSV-Endo-treated flank tumors completely sloughed. Immunohistochemical staining of the tumors revealed a decreased number of blood vessels in the HSV-Endo-treated group versus the control group. CONCLUSIONS: The oncolytic HSV-1 mutant HSV-Endo provided a two-pronged therapy; namely, inhibition of angiogenesis and direct tumor cell destruction by viral replication.

Allantois↗
PubMed2004

Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.

Animals↗
PubMed1998

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

Adenosine Triphosphatases↗
PubMed1978

Simian virus 40 DNA replication in isolated replicating viral chromosomes.

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.

Cell Line↗
PubMed2001

Conserved, N-linked carbohydrates of human immunodeficiency virus type 1 gp41 are largely dispensable for viral replication.

The transmembrane subunit (TM) of human immunodeficiency virus type 1 (HIV-1) envelope protein contains four well-conserved sites for the attachment of N-linked carbohydrates. To study the contribution of these N-glycans to the function of TM, we systematically mutated the sites individually and in all combinations and measured the effects of each on viral replication in culture. The mutants were derived from SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope sequence that originated from a primary HIV-1 isolate. The attachment site mutants were generated by replacing the asparagine codon of each N-X-S/T motif with a glutamine codon. The mobilities of the variant transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that all four sites are utilized for carbohydrate attachment. Transfection of various cell lines with the resulting panel of mutant viral constructs revealed that the N-glycan attachment sites are largely dispensable for viral replication. Fourteen of the 15 mutants were replication competent, although the kinetics of replication varied depending on the mutant and the cell type. The four single mutants (g1, g2, g3, and g4) and all six double mutants (g12, g13, g14, g23, g24, and g34) replicated in both human and rhesus monkey T-cell lines, as well as in primary rhesus peripheral blood mononuclear cells. Three of the four triple mutants (g124, g134, and g234) replicated in all cell types tested. The triple mutant g123 replicated poorly in immortalized rhesus monkey T cells (221 cells) and did not replicate detectably in CEMx174 cells. However, at 3 weeks posttransfection of 221 cells, a variant of g123 emerged with a new N-glycan attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental virus. The quadruple mutant (g1234) did not replicate in any cell line tested, and the g1234 envelope protein was nonfunctional in a quantitative cell-cell fusion assay. The synthesis and processing of the quadruple mutant envelope protein appeared similar in transient assays to those of the parental SHIV-KB9 envelope. Given their high degree of conservation, the four N-linked carbohydrate attachment sites on the external domain of gp41 are surprisingly dispensable for viral replication. The viral variants described in this report should prove useful for investigation of the contribution of carbohydrate moieties on gp41 to recognition by antibodies, shielding from antibody-mediated neutralization, and structure-function relationships.

Amino Acid Sequence↗
PubMed2006

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication.

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.

Cell Line↗
PubMed2005

Two Sp1/Sp3 binding sites in the major immediate-early proximal enhancer of human cytomegalovirus have a significant role in viral replication.

We previously demonstrated that the major immediate early (MIE) proximal enhancer containing one GC box and the TATA box containing promoter are minimal elements required for transcription and viral replication in human fibroblast cells (H. Isomura, T. Tsurumi, M. F. Stinski, J. Virol. 78:12788-12799, 2004). After infection, the level of Sp1 increased while Sp3 remained constant. Here we report that either Sp1 or Sp3 transcription factors bind to the GC boxes located at approximately positions -55 and -75 relative to the transcription start site (+1). Both the Sp1 and Sp3 binding sites have a positive and synergistic effect on the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. There was little to no change in MIE transcription or viral replication for recombinant viruses with one or the other Sp1 or Sp3 binding site mutated. In contrast, mutation of both the Sp1 and Sp3 binding sites caused inefficient MIE transcription and viral replication. These data indicate that the Sp1 and Sp3 binding sites have a significant role in HCMV replication in human fibroblast cells.

Binding Sites↗
PubMed2006

[HIV controllers: a homogeneous group of HIV-1 infected patients with a spontaneous control of viral replication].

We identified a group of chronically HIV-1-infected patients in whom viral replication is spontaneously controlled without antiretroviral treatment. These patients account for less than 1% of HIV-infected patients. They are defined according to a virological definition: plasma viral load less than 400 copies RNA/ml in more than 90% of the samples tested and HIV positive serology known for more than 10 years. They should never have received antiretroviral therapy. The CD4 T cell count is not taken into account, which differenciate these patients from long-term non progressors. Fifteen patients HIV controllers have been studied in a preliminary work with two main results: i) the demonstration of a strong HIV-specific CD8 immune response and ii) the amount of viral DNA in PBMCs is very low and stable along the time. The aim of the ANRS EP36 study group is to precize the immunological and virological characteristics of these patients. First data seem to confirm an essential role of CD8 T cells to control viral replication, the absence of defective virus, and a normal susceptibility of the CD4 T cells from these patients to the infection with various HIV strains. This group of patients is homogeneous and its study supports physiopathological studies. To go further, a national survey of these patients has been set up to allow future study.

Acquired Immunodeficiency Syndrome↗
PubMed1999

The conserved core of human immunodeficiency virus type 1 Nef is essential for association with Lck and for enhanced viral replication in T-lymphocytes.

The Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. We report the functional interactions of the Nef proteins with Src kinases. The Nef proteins from HIV-1 and SIV bind to Lck as well as Hck, Lyn, and Fyn. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. In addition, we show that SIV and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not phosphorylate HIV-1 Nef. These data suggest that the association of Nef and Lck is central to the enhanced viral replication of HIV-1 and SIV in T-lymphocytes.

Base Sequence↗