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At least 163 records · Page 9Linked to original sources

PubMed1992

New hydroxyethylamine HIV protease inhibitors that suppress viral replication.

The synthesis of analogues of AcSerLeuAsn[Phe-HEA-Pro]IleValOMe (1, JG-365; where HEA stands for the hydroxyethylamine unit 2), a tight-binding inhibitor of HIVP, are reported. Systematic modification of the P3 and P3' regions of the inhibitors has led to smaller HIVP inhibitors that inhibit viral replication in HIV-infected and SIV-infected cell cultures. Six aliphatic and/or aromatic derivatives were prepared by replacing residues in the P3 regions of BocLeuAsn[Phe-HEA-Pro]IleValOMe. Aromatic side chains at P3 gave better inhibitors than aliphatic side chains. The better inhibitors in this series contained a beta-naphthylalanine or a biphenyl unit at P3. A second series of HIVP inhibitors were obtained by converting the P3 group into acyl groups. CbzAsn[Phe-HEA-Pro]IlePheOMe and Qua-Asn-[Phe-HEA-Pro]-Ile-Phe-OMe (where Qua = quinolin-2-ylcarbonyl) are potent HIVP inhibitors with Ki values equal to 1.0 and 0.1 nM, respectively. The inhibition constants were determined by using the continuous fluorometric assay developed by Toth and Marshall. The activities of the protease inhibitors for inhibition of SIV replication were determined in vitro using CEM x 174 cells. Inhibition of HIV infection was determined essentially as reported by Pauwels and co-workers. The anti-HIV assay was carried out in culture using CEM cells (a CD4+ lymphocyte line) infected with virus strain HTLV-IIIb with a multiplicity of infection of 0.1. Several analogues inhibited the cytopathic effect at concentrations of 0.1-0.8 microgram/mL. These results establish that good inhibitors of HIV protease that inhibit viral replication in infected lymphocytes in in vitro cell assays can be obtained from JG-365 when the AcSerLeu unit is replaced by aromatic acyl derivatives.

Amino Acid Sequence↗

PubMed1998

Functional roles of HIV accessory proteins for viral replication (review).

Numerous lentiviruses, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) of causative agents of human AIDS as representative, have been recently isolated from various species of primates. The fundamental and most prominent feature of the viruses is the presence of a number of accessory genes in their genomes. Extensive biological and biochemical studies have demonstrated that the accessory gene products are not essential for viral replication at least in certain types of cells. Quite surprisingly, some of these accessory proteins are absolutely non-essential in any types of cells so far examined. In this brief review, our systematic genetic studies on the importance of the accessory proteins of HIV-1 and HIV-2 for viral replication are described and discussed.

Animals↗

PubMed2005

Expansion and contraction of HIV-specific CD4 T cells with short bursts of viremia, but physical loss of the majority of these cells with sustained viral replication.

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-gamma-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7-CD45RA-) and functional properties (IFN-gamma+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.

Alleles↗

PubMed2000

Simian immunodeficiency virus (SIV) gag DNA-vaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection.

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.

Animals↗

PubMed2003

Cytopathogenicity of pestiviruses: cleavage of bovine viral diarrhea virus NS2-3 has to occur at a defined position to allow viral replication.

Despite of highly divergent genome organizations, the N terminus of nonstructural protein 3 (NS3) is highly conserved between cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains. Generation of NS3, often by NS2-3 cleavage, is a marker of cp BVDV. The significance of the cleavage site within NS2-3 for viral replication was addressed by the use of BVDV replicons. Our results demonstrate that elongation as well as truncation of NS3 strongly interfere with viral RNA replication. This finding strongly suggests that the observed conservation of the N terminus of NS3 between cp BVDV is caused by functional selection and not by the presence of a hotspot of recombination.

Amino Acid Sequence↗

PubMed1994

IL-10 is induced during HIV-1 infection and is capable of decreasing viral replication in human macrophages.

IL-10 has been shown to be capable of down-regulating several aspects of macrophage function. This study was undertaken to define the association between IL-10 and HIV-1 infection in human macrophages. Infection of macrophages with a monocytotropic strain of the human immunodeficiency virus, HIV-BaL, resulted in expression of IL-10 mRNA within 3 to 12 h after infection, as determined by the reverse transcriptase PCR. Biologically active IL-10 was detected in supernatants from HIV-1-infected macrophages as early as 12 h post-infection. The addition of human rIL-10 to HIV-1-infected macrophage cultures resulted in a significant decrease in the viral replication. In addition, exogenous IL-10 blocked the ability of TNF-alpha to elevate viral replication. To determine whether IL-10 was associated with in vivo infection, lymph nodes from AIDS patients were examined for the presence of IL-10 mRNA by using PCR. IL-10 mRNA was evident in all lymph node tissue examined, but was absent in normal lymph node biopsies. These in vitro and in vivo findings demonstrate a strong and heterogeneous association between HIV-1 infection and IL-10.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Increased expression of toll-like receptor 2 on monocytes in HIV infection: possible roles in inflammation and viral replication.

BACKGROUND: Toll-like receptors (TLRs) are key pattern-recognition receptors of the innate immune system, but their role in human immunodeficiency virus (HIV) infection is largely unknown. METHODS: In the present study, we examined the expression of TLR2 and TLR4 on monocytes from 48 HIV-infected patients and 21 healthy control subjects by flow cytometry. RESULTS: We found that freshly isolated monocytes from HIV-infected patients displayed enhanced expression of TLR2 but not TLR4, that TLR2 expression on the surface of monocytes was significantly increased upon stimulation of HIV type 1 envelope protein gp120, and that TLR2 stimulation in HIV-infected patients induced increased viral replication and TNF- alpha response. CONCLUSION: Our findings suggest potential roles for TLR2 in chronic immune activation and viral replication in HIV infection.

Adult↗

PubMed1996

Inhibition of viral replication reverses respiratory syncytial virus-induced NF-kappaB activation and interleukin-8 gene expression in A549 cells.

Previous studies have demonstrated that respiratory syncytial virus (RSV) infection of airway epithelial cells results in the expression of a number of cytokines, such as interleukin-8 (IL-8), that are transcriptionally regulated by nuclear factor kappaB (NF-kappaB). In the studies reported here, we demonstrate that treatment of RSV-infected A549 cells with 100 microg of ribavirin (a viral replication inhibitor) per ml results in reversal of RSV-induced NF-kappaB activation, IL-8 mRNA expression, and IL-8 protein production in A549 cells. These data confirm that viral replication is a key step in RSV-induced NF-kappaB activation and IL-8 production.

Antiviral Agents↗

PubMed1989

Evidence of viral replication in HBsAg positive patients with hepatocellular carcinoma: measurement of serum hepatitis B virus deoxyribonucleic acid (HBV-DNA).

Sera from 60 consecutive patients with hepatocellular carcinoma (HCC) diagnosed by either a positive histology or at least two of the following: CT scan, hepatic ultrasound, hepatic angiogram and raised alpha-foetoprotein levels, were studied to determine their Hepatitis B surface antigen (HBsAg) status and the frequency of continuing viral replication. Markers of Hepatitis B virus (HBV) infection were detected using enzyme and radioimmunoassays. Hepatitis B DNA (HBV-DNA) was measured by molecular hybridization using a labelled recombinant HBV-DNA probe. Sera from 30 HBsAg positive 'healthy' carriers were used as controls. Forty-seven (78%) patients were HBsAg positive. Evidence of previous HBV infection was present in 12 of 13 HBsAg negative patients. 30% of HBsAg positive patients were HBeAg positive, 62% were anti-HBe positive and 8% had no 'e' markers. Serum HBV-DNA was present in 21 (45%) HBsAg positive patients: 10 (71%) HBeAg positive patients, 10 (35%) anti-HBe positive patients and 1 (25%) patient without any 'e' markers. Serum HBV-DNA levels were less than 0.6 ng/ml in 16 (76%) patients. HBV-DNA was absent in all HCC patients who were HBsAg negative. The frequency of serum HBV-DNA positivity was higher in 30 patients with HCC when compared with 30 age and sex matched healthy HBsAg positive individuals (57% vs 13% p less than 0.01). A sizeable proportion of HBsAg positive HCC patients have on-going viral replication and this is present in patients who were thought to have stopped replicating (anti-HBe positive patients).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2000

Human immunodeficiency virus type 1 pathogenesis in SCID-hu mice correlates with syncytium-inducing phenotype and viral replication.

Human immunodeficiency virus type 1 (HIV-1) patient isolates and molecular clones were used to analyze the determinants responsible for human CD4(+) thymocyte depletion in SCID-hu mice. Non-syncytium-inducing, R5 or R3R5 HIV-1 isolates from asymptomatic infected people showed little or no human CD4(+) thymocyte depletion in SCID-hu mice, while syncytium-inducing (SI), R5X4 or R3R5X4 HIV-1 isolates from the same individuals, isolated just prior to the onset of AIDS, rapidly and efficiently eliminated CD4-bearing human thymocytes. We have mapped the ability of one SI HIV-1 isolate to eliminate CD4(+) human cells in SCID-hu mice to a region of the env gene including the three most amino-terminal variable regions (V1 to V3). We find that for all of the HIV-1 isolates that we studied, a nonlinear relationship exists between viral replication and the depletion of CD4(+) cells. This relationship can best be described mathematically with a Hill-type plot indicating that a threshold level of viral replication, at which cytopathic effects begin to be seen, exists for HIV-1 infection of thymus/liver grafts in SCID-hu mice. This threshold level is 1 copy of viral DNA for every 11 cells (95% confidence interval = 1 copy of HIV-1 per 67 cells to 1 copy per 4 cells). Furthermore, while SI viruses more frequently achieve this level of replication, replication above this threshold level correlates best with cytopathic effects in this model system. We used GHOST cells to map the coreceptor specificity and relative entry efficiency of these early- and late-stage patient isolates of HIV-1. Our studies show that coreceptor specificity and entry efficiency are critical determinants of HIV-1 pathogenesis in vivo.

Amino Acid Sequence↗

PubMedDate not supplied

Treatment of human monocyte-derived macrophages with a TNF alpha synthesis inhibitor prior to HIV1 infection: consequences on cytokine production and viral replication.

Human monocyte-derived macrophages (MDM) were infected with the viral strain HIV1/Ba-L and with the clinical isolates HIV1/DAS and HIV1/PAR. Kinetics of tumour necrosis factor alpha (TNF alpha) and interleukin-6 (IL6) production were investigated for 28 days after infection. At the early stages of infection we observed significant TNF alpha and IL6 secretion 2 to 10 h after infection, whatever the viral strain we used. During the late events of MDM infection, TNF alpha and IL6 were detected over 16 to 21 days following HIV1 infection, at the time of high viral replication. Pretreatment of MDM with a TNF alpha synthesis inhibitor, RP 55778, 4 h prior to HIV infection induced a modified cytokine pattern during the first ten hours of infection: TNF alpha production was totally inhibited despite comparable amounts of IL6. At the late phases of the cell culture, a decrease in magnitude of both viral and cytokine production as well as a delay in the appearance of reverse transcriptase activity and cytokine secretion peaks were observed in RP-55778-pretreated and HIV1-infected MDM cultures. Similar results were obtained after pretreatment of HIV1/DAS-infected MDM cultures with an anti-TNF alpha monoclonal antibody.

Antibodies, Monoclonal↗

PubMed1990

A DNA ligase gene in the Copenhagen strain of vaccinia virus is nonessential for viral replication and recombination.

Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.

Amino Acid Sequence↗

PubMed2002

[Cloning, expression of the gI gene of pseudorabies virus ea strain and its effection to viral replication].

The complete gI was cloned from the genomic DNA of Pseudorabies virus (PRV) Ea strain and the DNA sequence was determined by Sanger's sequencing technique. The nucleotide and deduced animo acids sequences indicated that the gI gene of PRV Ea strain is composed of 1101 base pairs and could encode 366 animo acids residues. The result of predicted II class structure showed that gI is a typical I class envelope protein. When compared with PRV Rice strain, there were multiple mutations in the gI gene of PRV Ea strain, and the diversity of amino acid residues also existed, especially the deletion of two bases which results in the change of the reading frame and there were additional 16 animo acids residues in the potential cytoplasmic domains of Ea strain. The fragment containing the complete gI gene was further sub-cloned into the downstream of CMV promoter of eukaryotic expression vector pcDNA3.1+, resulting in a recombinant expression plasmid pSB209. pSB209 was transfected into PK-15 cell lines and the cells expressing gI were obtained in the selection of G418. Expressed protein was further detected by Indirect Immunofluorescence Assay(IFA). In order to explore the function of gI in the viral replication, the attenuated live vaccine strain, Bartha in which the gI is deleted, was chosen to determine the plaques forming units (pfu) and the tissue culture infectious dose(TCID50) in PK-15 cell lines expressing the gI(PKgI). The data showed that the pfu and TCID50 in PKgI were 164% and 200% of those in control cell lines respectively. The above results indicated that the gI involve in and accelerate the viral replication.

Amino Acid Sequence↗

PubMed1991

Cytokine influence on simian immunodeficiency virus replication within primary macrophages. TNF-alpha, but not GMCSF, enhances viral replication on a per-cell basis.

The control of HIV-1 or SIV replication within macrophages is probably influenced by a variety of viral and cellular factors. Of the cellular factors, the authors have studied cytokine influence on SIV replication in vitro utilizing simian alveolar macrophages and uncloned SIVmacMTV, a macrophage-tropic variant. The approach allowed quantification of viral replication on a per-cell basis. As reported for HIV-1 replication in macrophages, TNF-alpha significantly increased SIV production in these macrophage cultures. GMCSF also resulted in marked increases in SIV gag protein in culture supernatants. However, after correcting for differences in total cell numbers and numbers of gag-containing cells in the treated and untreated cultures, GMCSF did not upregulate SIV production on a per-cell basis. IL-6 increased SIV replication little if at all but induced significantly greater cytopathic changes in the treated cultures compared with infected, untreated cultures. In contrast, IFN-gamma greatly decreased replication. Our results for GMCSF, IFN-gamma, and IL-6 are in contrast to previously published reports of cytokine control of HIV-1 growth in target cells, and they stress the importance of cell number analyses and the use of primary cultures in the study of lentiviral replication kinetics in macrophages.

Animals↗

PubMed2006

Activation of focal adhesion kinase by hepatitis B virus HBx protein: multiple functions in viral replication.

The hepatitis B virus (HBV) X protein (HBx) is a multifunctional regulator of cellular signal transduction and transcription pathways and has a critical role in HBV replication. Much of the cytoplasmic signal transduction activity associated with HBx expression and its stimulation of viral replication is attributable to HBx-induced activation of calcium signaling pathways involving Pyk2 and Src tyrosine kinases. To further characterize upstream signal transduction pathways that are required for HBx activity, including activation of Src and mitogen-activated protein kinase (MAPK) cascades, we determined whether focal adhesion kinase (FAK), a known regulator of Src family kinases and the other member of the Pyk2/FAK kinase family, is activated by HBx. We report that HBx activates FAK and that FAK activation is important for multiple HBx functions. Dominant inhibiting forms of FAK blocked HBx activation of Src kinases and downstream signal transduction, HBx stimulation of NF-kappaB and AP-1-dependent transcription, and HBV DNA replication. We also demonstrate that HBx-induced activation of FAK is dependent on cellular calcium signaling, which is modulated by HBx. Moreover, prolonged expression of HBx increases both FAK activity and its level of expression. FAK activation may play a role in cellular transformation and cancer progression. HBx stimulation of FAK activity and abundance may also be relevant as a potential cofactor in HBV-associated hepatocellular carcinoma.

Animals↗

PubMed2005

HBeAg negative serological status and low viral replication levels characterize chronic hepatitis B virus-infected women at reproductive age in Greece: a one-year prospective single center study.

AIM: To evaluate the seroprevalence of hepatitis B surface antigen (HBsAg) in 13 581 women at reproductive age and the hepatitis B e antigen (HBeAg)/anti-HBe status as well as serum hepatitis B virus (HBV)-DNA levels in a subgroup of HBsAg(+) pregnant women at labor in Greece. METHODS: Serological markers were detected using enzyme immunoassays. Serum HBV-DNA was determined by a sensitive quantitative PCR assay. Statistical analysis of data was based on parametric methodology. RESULTS: Overall, 1.156% of women were HBsAg(+) and the majority of them (71.3%) were Albanian. The prevalence of HBsAg was 5.1% in Albanian women, 4.2% in Asian women and 1.14% in women from Eastern European countries. The prevalence of HBsAg in African (0.36%) and Greek women (0.29%) was very low. Only 4.45% of HBsAg(+) women were also HBeAg(+) whereas the vast majority of them were HBeAg(-)/anti-HBe(+). Undetectable levels of viremia (<200 copies/mL) were observed in 32.26% of pregnant women at labor and 29.03% exhibited extremely low levels of viral replication (<400 copies/mL). Only two pregnant women exhibited extremely high serum HBV-DNA levels (>10 000 000 copies/mL), whereas 32.26% exhibited HBV-DNA levels between 1 500 and 40 000 copies/mL. CONCLUSION: The overall prevalence of HBsAg is relatively low among women at reproductive age in Greece but is higher enough among specific populations. The HBeAg(-)/anti-HBe(+) serological status and the extremely low or even undetectable viral replicative status in the majority of HBsAg(+) women of our study population, suggest that only a small proportion of HBsAg(+) women in Greece exhibit a high risk for vertical transmission of the infection.

Adolescent↗

PubMed2001

Reactivation of viral replication after liver resection in patients infected with hepatitis B virus.

OBJECTIVE: To investigate the mechanisms and risk factors underlying postoperative reactivation of hepatitis after liver resection for hepatitis B virus-related hepatocellular carcinoma. SUMMARY BACKGROUND DATA: Although risk factors for acute hepatic failure after liver resection have been reported in patients with chronic liver disease, the issue of reactivation of hepatitis B virus replication after liver resection is unresolved. METHODS: Fifty-five patients with hepatocellular carcinoma and hepatitis B surface antigen underwent liver resection. In 25 of these 55 patients, serum levels of hepatitis B virus DNA and the type of hepatitis B virus were determined before and after surgery. RESULTS: Postoperative hepatitis occurred in 13 of the 55 patients (24%). Reactivation of viral replication occurred after liver resection in 7 of the 25 patients tested, and alanine aminotransferase activity increased in 6 of these 7 patients. High preoperative alanine aminotransferase activity, high levels of hepatitis B virus DNA, presence of wild-type DNA, and detection of hepatitis B core antigen in hepatocytes, all features of the immune clearance phase in the natural course of hepatitis B virus infection with no surgery, were more likely to be found in patients with reactivation than in patients without reactivation. CONCLUSIONS: During the immune clearance phase of hepatitis B virus infection, especially the period of acute exacerbation, changes in serum hepatitis B virus DNA level should be monitored for early warnings of reactivation of viral replication, likely to cause severe postoperative hepatitis and acute hepatic failure.

Adult↗

PubMed1994

Evidence that the herpes simplex virus type 1 uracil DNA glycosylase is required for efficient viral replication and latency in the murine nervous system.

Herpes simplex virus (HSV) encodes a uracil DNA glycosylase (UNG; UL2), which has been shown to be dispensable for normal replication of HSV-1 in cultured cells (J. Mullaney, H.W. Moss, and D.J. McGeoch, J. Gen. Virol. 70:449-454, 1989). In adult neurons, UNG activity is undetectable (F. Focher, P. Mazzarello, A. Verri, U. Hubscher, and S. Spadari, Mutat. Res. 237:65-73, 1990), suggesting that the HSV-1 UNG may play an important role in viral replication in neurons acutely and/or following reactivation. To examine the contribution of the HSV-1 UNG in vivo, two independent strain 17 Syn+ Ung- mutants, designated uB1 and uB2, were examined in a mouse model of herpetic disease. Following direct intracranial inoculation, both mutants exhibited a 10-fold reduction in neurovirulence compared with the parental strain 17 Syn+. Inoculations by a peripheral route demonstrated that the Ung- mutants were at least 100,000-fold less neuroinvasive than 17 Syn+. Replication kinetics in vivo demonstrated that uB1 and uB2 replicated less well in both the mouse peripheral and central nervous systems. Latency was established by both of the mutants in 100% of the animals examined. Following transient hyperthermia, however, the frequency of reactivation of the mutants in vivo was dramatically reduced. Restoration of the UNG locus resulted in full neurovirulence, neuroinvasiveness, and the ability to reactivate in vivo. These findings suggest that the HSV-1 UNG plays an important role during acute viral replication in vivo and possibly in the reactivation process.

3T3 Cells↗