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At least 937 records · Page 52Linked to original sources
PubMed1986

Effects of adenine arabinoside and corticosteroid on replication of duck hepatitis B virus DNA in the liver.

Adenine arabinoside (Ara-A) therapy and abrupt withdrawal of corticosteroids have both been used in the treatment of chronic infections due to hepatitis B virus (HBV). In order to better understand the effects and mechanism of action of these treatments, we treated ducks chronically infected with duck hepatitis B virus (DHBV) with different dosage regimens of the two therapies. We measured endogenous DNA polymerase activity and used sensitive molecular biological techniques to monitor serum and intrahepatic viral replicative forms during and after drug treatment. Ara-A had a transient, dose related inhibitory effect on DHBV replication. Viral plus strand synthesis was disproportionately affected. Following the cessation of Ara-A treatment markers of viral replication returned to their baseline values. We conclude that Ara-A exerts its effect through inhibition of viral DNA polymerase. Corticosteroid treatment results in an increase in DHBV replication, but steroid withdrawal results in a short-lived transient decrease in markers of viral replication to below pretreatment values. Our results suggest that steroid withdrawal decreases hepadna virus replication through a mechanisms of immune modulation. On the basis of these results and previous trials in HBV infected patients, we predict that neither agent will efficiently eliminate viral replication in chronic hepadna virus infection when used as the sole therapeutic modality. We suggest that the differences in the mechanisms of action of Ara-A treatment and corticosteroid withdrawal be exploited, and the use of combination therapy be explored.

Adrenal Cortex Hormones↗
PubMed1991

In vitro deletion mapping of the viral strand replication origin of Pseudomonas bacteriophage Pf3.

The origin of viral strand replication of the filamentous bacteriophage Pf3 has been characterized in Escherichia coli by in vitro deletion mapping techniques. The origin region was functionally identified by its ability to convey replicative properties to a recombinant plasmid in a polA host in which the replication origin of the vector plasmid is not functional. The origin of Pf3 viral strand replication is contained within a DNA sequence of 139 bp. This sequence covers almost completely one of the intergenic regions of the Pf3 genome, and it specifies both replication initiation and termination functions. Although no nucleotide sequence homology is present between the Pf3 origin of viral strand replication and that of the E. coli filamentous phages Ff (M13, f1, and fd) and IKe, their map positions and functional properties are very similar.

Bacteriophages↗
PubMed2005

TATA-binding protein and TBP-associated factors during herpes simplex virus type 1 infection: localization at viral DNA replication sites.

Host RNA polymerase II (RNAP II) is responsible for viral transcription of the herpes simplex virus type 1 (HSV-1) genome and is relocalized to viral DNA replication compartments. Thus, we investigated whether TATA-binding protein (TBP) and TBP-associated factors (TAFs) are recruited to sites of viral transcription and replication and whether TBP/TAF expressions are influenced upon infection. The protein levels of TBP, hsTAF1/TAF(II)250, hsTAF4/TAF(II)135, and hsTAF5/TAF(II)100 were constant during the early phase of infection and started to decrease late during infection. Only for hsTAF7/TAF(II)55 we sometimes observed a decrease already at 4-8h postinfection (p.i.). Concomitantly with the relocalization of RNAP II, TBP and hsTAFs were redistributed to sites of viral DNA replication and transcription. In the absence of viral DNA replication TBP/hsTAFs were present in distinct nuclear dots, however, enlargement of the nuclear structures did not take place. Our results show that HSV-1 infection has no influence on the protein levels of TFIID components and leads to a redistribution of TBP and hsTAFs to prereplicative sites that enlarge to viral DNA replication compartments.

Cell Nucleus↗
PubMed1996

Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication.

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.

Animals↗
PubMed1993

High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV-1-infected chimpanzees.

We have examined human immunodeficiency virus type 1 (HIV-1) infection in chimpanzees by analyzing HIV-1 DNA and RNA in lymph nodes and peripheral mononuclear cells (PBMCs). Like certain asymptomatic HIV-infected persons, these chimpanzees had no detectable viral replication in their PBMCs. However, viral replication and a high viral load were observed in the lymphatic tissue. Despite the absence of viral replication in PBMCs, 1/1,000 to 1/10,000 of the PBMCs contained HIV-1 proviral DNA, and HIV transcription could be rapidly induced in these cells in vitro. These results provide direct evidence of cellular latency of HIV in vivo and suggest that HIV infection in chimpanzees may be a useful model for clinical latency of HIV infection in humans.

Animals↗
PubMed1991

Simultaneous detection of highly phosphorylated proteins and viral major DNA binding protein distribution in nuclei of adenovirus type 5-infected HeLa cells.

Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism.

Adenoviruses, Human↗
PubMed1988

Expression of preS1 and preS2 in the liver of chronic hepatitis B virus carriers.

Using monoclonal antibodies we have studied the expression and distribution pattern of preS1 and preS2 proteins in the liver of 25 patients with chronic hepatitis B virus infection. Both preS1 and preS2 were detected in the liver of most cases, independently of viral replication and of the state of the viral genome in the liver. In fact, preS1 and preS2 were present in the liver of 14 out of 15 patients with HBV-DNA in the serum as well as of 7 out of 10 cases with no evidence of viral replication, including 2 with integrated HBV-DNA sequences in the liver and 5 patients with no evidence of liver damage. The pattern of distribution of preS proteins was identical to that of hepatitis B surface antigen, with exclusive cytoplasmic staining in cases without viral replication and cytoplasmic and membranous positivity in those with viral replication. These results clearly indicate that translation of preS proteins is independent of viral replication and is not necessarily associated with liver damage.

Antibodies, Monoclonal↗
PubMedDate not supplied

Repression of simian virus 40 early transcription by viral DNA replication in human 293 cells.

The small DNA tumour virus simian virus 40 (SV40) has served as an excellent model for many studies on the mechanism and control of gene expression in eukaryotic cells. The SV40 early region produces two protein products. One product (large-T antigen) is known both to repress early viral transcription and to stimulate viral replication by binding to specific sites in the origin-promoter region. The early promoter has several similarities to other RNA polymerase II promoters, for example, it possesses a TATA box, an upstream element and an enhancer. However, the SV40 early promoter differs from other known RNA polymerase II promoters in that the origin of viral DNA replication is embedded within it. Here we show that the SV40 early region is expressed at an extremely low level following its introduction ito human 293 cells, contrasting with results observed in a large number of other cells lines. We show further that the lack of expression is due to repression of transcription from the SV40 early promoter by viral DNA replication which occurs efficiently in 293 cells.

Adenoviridae↗
PubMed2002

Biochemical and biological characterization of a dodecameric CD4-Ig fusion protein: implications for therapeutic and vaccine strategies.

Drug toxicities associated with HAART lend urgency to the development of new anti-HIV therapies. Inhibition of viral replication at the entry stage of the viral life cycle is an attractive strategy because it prevents de novo infection. Soluble CD4 (sCD4), the first drug in this class, failed to suppress viral replication in vivo. At least three factors contributed to this failure: sCD4 demonstrated poor neutralizing activity against most primary isolates of HIV in vitro; it demonstrated an intrinsic capacity to enhance viral replication at low concentrations; and it exhibited a relatively short half-life in vivo. Many anti-gp120 monoclonal antibodies, including neutralizing monoclonal antibodies also enhance viral replication at suboptimal concentrations. Advances in our understanding of the events leading up to viral entry suggest strategies by which this activity can be diminished. We hypothesized that by constructing a sCD4-based molecule that is large, binds multiple gp120s simultaneously, and is highly avid toward gp120, we could remove its capacity to enhance viral entry. Here we describe the construction of a polymeric CD4-IgG1 fusion protein. The hydrodynamic radius of this molecule is approximately 12 nm. It can bind at least 10 gp120 subunits with binding kinetics that suggest a highly avid interaction toward virion-associated envelope. This protein does not enhance viral replication at suboptimal concentrations. These observations may aid in the design of new therapeutics and vaccines.

AIDS Vaccines↗
PubMed1971

Evidence for and localization of vegetative viral DNA replication by autoradiographic detection of RNA-DNA hybrids in sections of tumors induced by Shope papilloma virus.

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis.

Animals↗
PubMed2005

Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.

Amino Acid Sequence↗
PubMed2002

Viral evolution and challenges in the development of HIV vaccines.

Potent virus-specific cytotoxic T lymphocyte (CTL) responses elicited by candidate AIDS vaccines have been shown to provide short-term control of viral replication following pathogenic viral challenges in rhesus monkeys. We have recently shown that vaccines that control rather than prevent immunodeficiency virus infections are still subject to immune escape. In particular, viral mutations can develop that result in viral escape from recognition by immunodominant CTL, loss of immune control of viral replication, and clinical disease progression. These data suggest that viral escape from CTL may prove to be a significant limitation of the current generation of CTL-based AIDS vaccines.

AIDS Vaccines↗
PubMed1998

Active domains of human papillomavirus type 11 E1 protein for origin replication.

Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.

Binding Sites↗
PubMed2003

HIV-1 replication increases HIV-specific CD4+ T cell frequencies but limits proliferative capacity in chronically infected children.

This study investigated the relationship between HIV-1 replication and virus (HIV-1; CMV)-specific CD4(+) T cell frequency and function in HIV-1-infected children. HIV-1 gag p55-specific CD4(+) T cell IFN-gamma responses were detected in the majority of children studied. p55-specific responses were detected less commonly and at lower frequencies in children with <50 copies/ml plasma HIV-1 RNA than in children with active HIV-1 replication. In children with <50 copies/ml plasma HIV-1, p55-specific responses were detected only in children with evidence of ongoing HIV-1 replication, indicating a direct relationship between HIV-1 replication and HIV-specific CD4(+) T cell frequencies. In contrast, p55-specific proliferative responses were detected more frequently in children with <50 copies/ml plasma HIV-1. CMV-specific CD4(+) responses were more commonly detected and at higher frequencies in CMV-coinfected children with suppressed HIV-1 replication. The lack of HIV-specific CD4(+) proliferative responses, along with the preservation of CMV-specific CD4(+) responses in children with controlled HIV-1 replication, suggests that viral replication may have deleterious effects on HIV-1 and other virus-specific CD4(+) responses. Vaccination to stimulate HIV-specific CD4(+) T cell responses in these children may synergize with antiretroviral therapy to improve the long-term control of viral replication, and may perhaps allow the eventual discontinuation of antiretroviral therapy.

Adolescent↗
PubMed2003

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication.

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes.

Base Sequence↗