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Results for “viral replication”

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PubMed1988

Tissue tropism and target cells of bluetongue virus in the chicken embryo.

In situ cytohybridization was used to determine the tissue tropism and target cells for replication of bluetongue virus (BTV) in the developing chicken embryo. Hybridization with a biotinylated probe specific for segment 3 of BTV serotype 17 detected viral replication in embryos inoculated with U.S. serotypes 2, 10, 11, 13, and 17 or sheep blood containing a BTV field strain. At the final stages of infection, when the embryos were hemorrhagic, viral infection could consistently be detected in the brain, kidney, spinal cord, heart, lung, and liver, with the brain and kidney most severely affected. Other tissues, such as the retina, skin, tongue, and intestinal villi, also supported viral replication in some embryos. Greater concentrations of virus tended to be localized within epithelial cells, such as those lining the kidney tubules and tertiary bronchi of the lungs. Kinetics studies with BTV serotypes 11 and 17 and a field strain indicated that within 24 h after inoculation, viral replication occurred initially in the brain and kidney. By 48 h, viral replication was also detected in the lungs, heart, and spinal cord, with the liver being severely infected by 72 h. Low levels of hybridization could be detected in embryos infected with epizootic hemorrhagic disease virus, which is antigenically related to BTV.

Animals↗

PubMed1987

A new 'sense' RNA approach to block viral RNA replication in vitro.

The use of "antisense" RNA is being widely considered to block specific steps in viral infection. We propose here a new "sense" RNA approach to block viral RNA replication in vitro and possibly in vivo. In the turnip yellow mosaic virus (TYMV) system, the recognition site of the viral replicase (RNA-dependent RNA polymerase) is assumed to be located within the 3' end of the RNA genome. Small "sense" RNAs have been obtained by in vitro transcription of the corresponding cloned cDNAs. Replication of TYMV RNA in vitro is shown here to be blocked only by those RNAs that contain the 3' terminal region of the genome.

Antiviral Agents↗

PubMed1993

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells.

Cytosine↗

PubMed2004

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.

The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex.

Genotype↗

PubMed2005

An inhibitor of c-Jun N-terminal kinases (CEP-11004) counteracts the anti-HIV-1 action of trichosanthin.

Trichosanthin (TCS) is a type I ribosome-inactivating protein possessing multiple biological and pharmacological activities. One of its major actions is inhibition of human immunodeficiency virus (HIV) replication. The mechanism is still not clear. It is generally believed that this action is mediated via ribosome inactivation. Recently, we found that some TCS mutants with full ribosome inactivating activity were devoid of anti-HIV-1 effect. This suggested that there might be other mechanisms contributing to the anti-HIV-1 action. This study showed that a commonly used c-Jun N-terminal kinases inhibitor (CEP-11004) could counteract the antiviral action of TCS in C8166 cells. CEP-11004 alone had no effect on HIV-1 replication and TCS alone significantly inhibited this process. When CEP-11004 was used together with TCS, the antiviral action of TCS was much reduced. Two methods were used to assess viral replication. (1) By measuring the HIV-1 reverse transcriptase, TCS on the average reduced viral replication to 52+/-4%. With CEP-11004 pretreatment, TCS appeared to lose the HIV-1 inhibitory activity with viral replication stood at 101+/-7%. (2) By measuring HIV-1 p24, TCS reduced viral replication to 68+/-4%. With CEP-11004 pretreatment, TCS again seemed to lose its anti-HIV-1 activity with HIV-1 replication rose back to 101+/-4%. Both indexes indicated that CEP-11004 counteracted the antiviral action of TCS. Phosphorylation of JNK on the other hand was only slightly elevated by 1.5-fold by TCS and CEP-11004 inhibited this elevation. These results suggested that the anti-HIV-1 effect of TCS may be related to the MAPK signal process downstream from the point of CEP inhibition.

Anti-HIV Agents↗

PubMed1997

Identification of the functional regions required for hepatitis D virus replication and transcription by linker-scanning mutagenesis of viral genome.

To define the important cis-elements in hepatitis delta virus (HDV) RNA, the viral genome was mutated by a linker-scanning mutagenesis strategy that maintained the native rod-like structure of HDV RNA. Mutant HDV cDNAs or their corresponding RNA transcripts were transfected into a Huh-7-derived cell line which continuously expressed small hepatitis delta antigen to study the viral replication and transcription. Here we report the following findings. (i) Although most of the mutant RNAs could self-process to generate the 1.7-kb genomic RNA and all their stabilities were similar, positions which surround the genomic ribozyme domain were found to be important for the self-processing of the dimeric RNA. (ii) The replication of viral RNA was greatly diminished in many mutants, suggesting that multiple regions in HDV RNA were required for replication. (iii) In certain mutants, replication of the HDV antigenomic RNA was selectively abolished but that of the genomic RNA was not. Therefore, this was the first report to show that the cis-elements needed for the replication of genomic or antigenomic HDV RNA could be different. (iv) A continuous region (nt 1625 to nt 431), spanning the HDAg mRNA initiation site and containing the in vitro identified RNA promoter, was found to be important for mRNA production in vivo. (v) The HDV RNA replication and transcription was previously proposed to be governed by a single "double-acting promoter." However, two mutants which were deficient in mRNA synthesis still retained active viral RNA replication. It suggested that the HDV replication could initiate from sites other than this single promoter. This study therefore provided an insight into the cis-elements required for HDV RNA replication and transcription and further contributed to our understanding of HDV life cycle.

Genome, Viral↗

PubMed2001

HIV-1 integration as a target for antiretroviral therapy: a review.

Since the discovery of the human immunodeficiency virus type 1 (HIV-1) as the causative agent of AIDS in the early eighties, its spread has been dramatic. Current therapeutic strategies for the inhibition of viral replication employ a combination of drugs targeted at the viral reverse transcriptase and protease enzymes. The clinical benefit of this combination therapy is considerable, although often only transient, partly due to the emergence of multiple drug-resistant viral strains. The addition of new anti-HIV drugs targeting a third step of the viral replication may help in preventing resistance development. During HIV replication, the integration of the genome into the cellular chromosome is a vital step, which is catalysed by the viral integrase. The search for antiviral compounds capable of selective inhibition of integrase during viral replication is laborious and the large-scale screening programs for integrase inhibitors have thus far led to only one series of compounds that selectively inhibit the integration step of HIV replication, the diketo acids. In this review we summarize the current knowledge about HIV-1 integrase and integrase inhibitors. We address the issue why it is so difficult to find potent and selective integrase inhibitors, suitable to be included in a therapeutic drug combination and we propose new strategies for the discovery of integration inhibitors.

Active Transport, Cell Nucleus↗

PubMed2005

Immunologic function and virologic suppression among children with perinatally acquired HIV Infection on highly active antiretroviral therapy.

BACKGROUND: The goal of highly active antiretroviral therapy (HAART) has been to stabilize and reconstitute immune function and suppress viral replication to the greatest degree possible. Suppression of HIV viral replication has been associated with improved long-term and short-term prognosis. Limited data are available on the level of virologic suppression and immune function of pediatric patients followed in clinical settings in the HAART era. OBJECTIVE: The objective of this study was to assess the level of virologic suppression and immune function in a cohort of children with perinatally acquired HIV infection followed at dedicated HIV specialty care sites. RESEARCH DESIGN: This study comprised a cohort study of HIV-infected children and adolescents. SUBJECTS: Study subjects consisted of 263 HIV-positive children ( 25; only 4.2% had CD4 <15%. Overall, 12.5% of patients had either CD4% <15 or severely decreased absolute CD4 counts (adjusted for age). A total of 4.6% of patients had HIV-1 RNAs >100,000 cpm and severe immunosuppression. Patients who were less likely to achieve virologic suppression to <400 cpm included those with CD4 count <200 cells/mm(3) (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.007-0.46), those with AIDS (OR, 0.5; 95% CI, 0.28-0.94), and those with moderate (OR, 0.42; 95% CI, 0.22-0.79), or severe immunologic suppression (OR, 0.14; 95% CI, 0.046-0.43) based on CD4%. CONCLUSION: In this multisite, pediatric cohort, the rate of near-complete virologic suppression (<50 or <400 cpm) was low. However, the majority of patients have near-normal CD4 counts and viral loads <15,000 cpm. Follow up will be critical to assess the implications of ongoing low-level viral replication with near-normal CD4 values.

Adolescent↗

PubMed1990

Activities and mechanisms of action of halogen-substituted flavanoids against poliovirus type 2 infection in vitro.

The effects of some halogen-substituted flavanoids (dichloroflavan, halogenated isoflavans, and isoflavenes) on poliovirus type 2 infection was examined. Only two isoflavenes exhibited a significant inhibitory activity on the virus-induced cytopathic effect and plaque formation. In a single cycle of viral replication, both compounds reduced the viral yield by approximately 90%. The presence of the isoflavenes from the beginning of infection or during the adsorption period only prevented the shutoff of host translation and viral RNA and protein synthesis, suggesting that the drugs blocked an early step of viral replication. Indeed, both isoflavenes were not virucidal, did not protect virus infectivity from heat inactivation, and had no measurable effect on the binding of virus to cells, viral penetration, and uncoating of the viral RNA. In contrast, both compounds significantly reduced the infectivity of free viral RNA. The possibility that compounds interfere with poliovirus replication at a very early stage of translation of the input RNA is discussed.

Flavonoids↗

PubMed1995

A conserved motif at the 3' end of mouse hepatitis virus genomic RNA required for host protein binding and viral RNA replication.

A conserved 11-nucleotide sequence, UGAAUGAAGUU, at the 3' end of the genomic RNA of coronavirus mouse hepatitis virus was required for host protein binding and viral RNA synthesis. An RNA probe containing this 11-nucleotide sequence bound four cellular proteins with a highly labeled protein of 120 kDa and three minor species with sizes of 103, 81, and 55 kDa. Mutation of the 11-nucleotide motif abolished cellular protein binding. The RNA-protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable from those observed with extracts from uninfected cells. Both negative-strand synthesis and positive-strand replication of viral defective interfering RNAs in the presence of helper virus were affected by mutations that disrupt RNA-protein complex formation, even though the 11 mutated nucleotides were converted to the wild-type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred relatively early in the MHV replicative cycle at 5.5 to 7.5 hr postinfection, a time when viral RNA synthesis and replication of positive-strand DI RNA were at barely detectable levels. A DI RNA with a mutation upstream of the protein binding element replicated as efficiently as wild type without undergoing recombination. Thus, the 11-nucleotide conserved host protein binding motif appears to play an important role in viral RNA replication.

Animals↗

PubMed1998

Characterization of age- and dose-related outcomes of duck hepatitis B virus infection.

Experimental inoculation of naive ducks with duck hepatitis B virus (DHBV) can lead to one of three outcomes, namely, persistent viremia, transient infection with or without viremia, or no evidence of infection. The ability of individual ducks to resolve DHBV infection was found to be linked to the age of the duck at the time of inoculation and the dose of inoculated virus. (1) In recently hatched ducks inoculated intravenously (i.v.) with 4 x 10(4) DHBV DNA genomes, a switch from persistent viremia to transient antibody appearance was seen at an age of inoculation between 7 and 14 days. A 25-fold increase in the dose of virus (1 x 10(6) DHBV genomes) delayed this switch by 7 days. (2) When 4-month-old ducks were inoculated i.v. with different doses of virus, only those receiving the highest dose (2 x 10(11) DHBV genomes) showed viremia and extensive viral replication and histological changes in the liver; 2/3 ducks in this group had a transient infection, while the third duck had viral replication and histological changes in the liver that were still present at day 120 postinoculation (p.i.). In all ducks receiving lower doses (1 x 10(3), 1 x 10(6), 1 x 10(9) DHBV genomes) antibodies to viral surface and core antigens developed without detectable viral replication in the liver on days 6, 9, or 12 p.i. (3) When 10- to 16-month-old ducks were inoculated i.v. with 2 x 10(11) DHBV genomes, all showed extensive viral replication in hepatocytes and mild to moderate histological changes in the liver on days 4 or 6 p.i. In 4/5 ducks viremia was not detected, anti-surface antibodies were first detected on day 8 p.i., and viral DNA and antigen were cleared from the liver by days 35-47 p.i. The remaining duck became viremic with persistence of virus in the liver until at least day 46 p.i. The findings of the study are consistent with a model for noncytopathic viruses (R. M. Zinkernagel (1996) Science 271, 173-178).

Age Factors↗

PubMed1967

Studies of the activity of diphtheria toxin. I. Poliovirus replication in intoxicated HeLa cells.

It has been demonstrated that a saturating dose of diphtheria toxin produced a 90% inhibition of polio-virus replication in HeLa cells. This inhibition was reflected in infectious viral RNA synthesis and in mature virus production. Toxin had no direct effect on virus particles or I-RNA, and poliovirus adsorption and eclipse appeared to be carried out normally in intoxicated cells. When toxin was given at various time intervals after infection, the amount of inhibition depended on the time of toxin addition. Toxin given before or immediately after infection gave maximum inhibition, while toxin given several hours after infection had little effect. The data suggest that toxin inhibits viral replication through its effect on protein synthesis. It is likely that a critical step in the viral replication cycle, the production of poliovirus-induced RNA polymerase, is inhibited, and possibly the synthesis of capsid protein. Ammonium salts and the aliphatic amines, glycamine and prolamine, prevented the inhibition of viral replication by toxin. The kinetics of the protective action of ammonium chloride and diphtheria antitoxin are remarkably similar.

Diphtheria Toxin↗

PubMed2006

Nonstructural protein of infectious bursal disease virus inhibits apoptosis at the early stage of virus infection.

Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). This protein has been implicated to play a role in the induction of apoptosis. In this study, we investigate the kinetics of viral replication during a single round of viral replication and examine the mechanism of IBDV-induced apoptosis. Our results show that it is caspase dependent and activates caspases 3 and 9. Nuclear factor kappa B (NF-kappaB) is also activated and is required for IBDV-induced apoptosis. The NF-kappaB inhibitor MG132 completely inhibited IBDV-induced DNA fragmentation, caspase 3 activation, and NF-kappaB activation. To study the function of the NS protein in this context, we generated the recombinant rGLS virus and an NS knockout mutant, rGLSNSdelta virus, using reverse genetics. Comparisons of the replication kinetics and markers for virally induced apoptosis indicated that the NS knockout mutant virus induces earlier and increased DNA fragmentation, caspase activity, and NF-kappaB activation. These results suggest that the NS protein has an antiapoptotic function at the early stage of virus infection.

Animals↗

PubMed1996

Role of reactive oxygen intermediates in cytomegalovirus gene expression and in the response of human smooth muscle cells to viral infection.

Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV-SMC interactions. Using confocal microscopy to identify a redox-sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated nuclear factor kappa B (NF kappa B), a cellular transcription factor, as demonstrated by increased NF kappa B binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NF kappa B activation. By using antioxidants to assess the role of ROIs in modulating virally mediated effects, we also found that CMV-induced ROIs (1) are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate-early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream from the MIEP) and (2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. Because ROIs, through activation of NF kappa B, can also induce expression of cellular genes involved in immune and inflammatory responses, the ROI response to CMV infection may also represent a parallel survival mechanism that has evolved in the host cell to protect against viral infection. We conclude that CMV induces intracellular ROI generation within minutes after infection of SMCs and then uses these ROIs to facilitate its own gene expression and replication. Conversely, antioxidants inhibit CMV immediate-early gene expression and viral replication.

Animals↗

PubMed2006

Interaction of macrophages with apoptotic cells enhances HIV Type 1 replication through PGE2, PAF, and vitronectin receptor.

Phagocytosis of apoptotic cells by macrophages increases secretion of soluble mediators and generates an antiinflammatory environment. We previously reported that phagocytosis of apoptotic cells by HIV-1-infected macrophages enhances viral replication, with the participation of the cytokine transforming growth factor- beta1 and an integrin receptor. Now, we describe the role of prostaglandin E2 (PGE2), platelet-activating factor (PAF), and the integrin alphaVbeta3 (vitronectin receptor, VnR) in this phenomenon. Exacerbation of HIV-1 growth induced by phagocytosis of apoptotic cells was inhibited when HIV-1-infected macrophages were treated with a cyclooxygenase 2 inhibitor, or with a PAF receptor antagonist (BN 52021) immediately after macrophage interaction with apoptotic cells. Treatment of HIV-1-infected macrophages with BN 52021 decreased viral replication, whereas addition of PGE2 or PAF to these cells enhanced viral replication. Monoclonal antibodies (MAbs) to VnR reduced the macrophage uptake of apoptotic cells, prevented the enhancement of HIV-1 growth upon the engulfment of apoptotic cells, and potently augmented viral replication in HIV-1-infected macrophages in the absence of apoptotic cells. In conclusion, PGE2 and PAF, and ligation of VnR as well, contribute to amplify viral growth in HIV-1-infected macrophages upon uptake of apoptotic cells.

Apoptosis↗

PubMed2005

Oncolytic adenoviruses - selective retargeting to tumor cells.

Virotherapy is an approach for the treatment of cancer, in which the replicating virus itself is the anticancer agent. Virotherapy exploits the lytic property of virus replication to kill tumor cells. As this approach relies on viral replication, the virus can self-amplify and spread in the tumor from an initial infection of only a few cells. The success of this approach is fundamentally based on the ability to deliver the replication-competent viral genome to target cells with a requisite level of efficiency. With virotherapy, while a number of transcriptional retargeting strategies have been utilized to restrict viral replication to tumor cells, this review will focus primarily on transductional retargeting strategies, whereby oncolytic viruses can be designed to selectively infect tumor cells. Using the adenoviral vector paradigm, there are three broad strategies useful for viral retargeting. One strategy uses heterologous retargeting ligands that are bispecific in that they bind both to the viral vector as well as to a cell surface target. A second strategy uses genetically modified viral vectors in which a cellular retargeting ligand is incorporated. A third strategy involves the construction of chimeric recombinant vectors, in which a capsid protein from one virus is exchanged for that of another. These transductional retargeting strategies have the potential for reducing deleterious side effects, and increasing the therapeutic index of virotherapeutic agents.

Adenoviridae↗

PubMed1999

Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis.

BACKGROUND: Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. METHODS AND RESULTS: We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). CONCLUSIONS: These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Adult↗

PubMed1977

Variations in integration site of avian oncornaviruses in different hosts.

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.

Animals↗