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PubMed2006

Human cytomegalovirus UL27 is not required for viral replication in human tissue implanted in SCID mice.

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.

Animals↗

PubMed1999

Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication.

Hepatitis B virus (HBV) core protein is a phosphoprotein. Its three major phosphorylation sites have been identified at the serine residues located at amino acids 157, 164, and 172. In order to investigate the role of core protein phosphorylation in HBV replication, these three serine residues were mutated to alanine to mimic nonphosphorylated serine or to glutamic acid to mimic phosphoserine. The nonphosphorylated core protein analog did not package the HBV pregenomic RNA, and the phosphorylated analog packaged the pregenomic RNA but failed to support viral DNA replication. These results indicate that the core protein phosphorylation may be important for pregenomic RNA packaging and that its dephosphorylation may be important for viral DNA replication. The individual roles of these three major phosphorylation sites in HBV replication were further investigated by being mutated to alanine in different combinations. The results showed that the serine residue at amino acid 157 was not essential for pregenomic RNA packaging, whereas the serine residues at amino acids 164 and 172 were more important. Furthermore, the serine residue at amino acid 157 was not essential for viral DNA replication or viral maturation.

Carcinoma, Hepatocellular↗

PubMedDate not supplied

Effects of IV cyclophosphamide on HIV viral replication in a patient with systemic lupus erythematosus.

Several cases of patients with concomitant SLE and HIV infection have been reported in the literature; however, the effect of immunosuppressive therapy on HIV replication has not been described. We present the case of a 46 y/o woman with a ten-year history of HIV infection who was treated with IV cyclophosphamide for SLE nephritis. She had a positive HIV Western Blot just a few months before the diagnosis of SLE. Serum levels of HIV RNA had been persistently non-detectable since the assay became available. The patient was not receiving any antiretroviral therapy, raising doubts about the diagnosis of HIV infection. After 3 pulses of IV cyclophosphamide, HIV RNA levels went up to 135,720 copies/ml. Shortly after discontinuation of therapy viral levels were again undetectable. This case shows one of the possible clinical scenarios in patients with coexistent HIV infection and SLE. In our patient SLE appears to provide some immunologic defense against viral replication. Cross-reactivity of autoantibodies with HIV proteins may play a role in this mechanism. Effective immunosuppressive therapy suppresses this protection and leaves the immune system vulnerable to HIV reproduction. Treatment in these cases can be difficult and should be individualized in an attempt to achieve a balance between control of viral infection and SLE activity.

CD4-Positive T-Lymphocytes↗

PubMed2004

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus.

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.

Adenosine Triphosphatases↗

PubMed1978

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

Adenoviruses, Human↗

PubMed2005

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo.

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.

Base Sequence↗

PubMed2005

CD16+ monocytes exposed to HIV promote highly efficient viral replication upon differentiation into macrophages and interaction with T cells.

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.

CD4 Antigens↗

PubMed1996

Stably expressed antisense RNA to cytomegalovirus UL83 inhibits viral replication.

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a phosphoprotein of 64 to 68kDa (pp65) which is a major constituent of this virion and dense bodies. To determine the importance of the HCMV gene in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL83 cDNA. Reverse transcription-PCR detected antisense RNA in the cytoplasm. The steady-state level of a 4-kb RNA containing coding sequences for pp65 was significantly reduced after infection of antisense cells. Concomitant with this, levels of expression of pp65 and pp71 (UL82) were severely reduced. Extracellular HCMV production was almost completely blocked, irrespective of the multiplicity of infection or the time after infection studied. The block occurred at an early phase, since immediate-early protein synthesis occurred normally, while several late proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were absent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequences of antisense RNA, demonstrated the specificity of the block for wild-type HCMV in the antisense-stabilized cells and indicated that the block was not due to indirect interference with cellular genes. Our results appear to contradict those of Schmolke et al (S. Schmolke, H.F. Kern, P. Drescher, G. Jahn, and B. Plachter, J. Virol. 69:5959-5968, 1995), which show that UL83 is a nonessential gene for HCMV replication in vitro. This contradiction is discussed in light of the fact that the 4-kb mRNA, which codes for pp65 and was targeted in UL83-antisense cell lines, may be a bicistronic mRNA which also codes for pp71 (UL82). Thus, interference of expression from the genes encoding pp65 and pp71 by blocking of this putative bicistronic message leads to severe impairment of viral replication.

Antiviral Agents↗

PubMed1975

Viral replication and interferon production in fetal and adult ovine leukocytes and spleen cells.

Peripheral blood leukocyte and spleen cell cultures derived from adult sheep and from third-trimester (107 to 145 days of gestation) and second-trimester (70 to 98 days of gestation) fetal lambs were examined for their ability to support viral replication and to produce interferon. Bluetongue virus, Herpesvirus hominis type 2, and Chikungunya virus failed to replicate in either leukocyte or spleen cell cultures derived from adult ewes or in cultures from second- or third-trimester fetal lambs. Similarly, peripheral blood leukocytes from adult sheep or third-trimester fetal lambs did not support the replication of Semliki Forest virus, vesicular stomatitis virus, Newcastle disease virus, or vaccinia virus. No major differences were observed in the ability of fetal and adult leukocytes to produce interferon in response to viral infection. In contrast, mean interferon titers induced by bluetongue virus, H. hominis type 2, and Chikungunya virus in spleen cells from second-trimester fetuses were 4- to 10-fold greater than those induced in spleen cells from adult ewes. Variations in interferon levels induced on separate occasions with cells from the same donor age group were observed. The antiviral substance induced in both the fetal and adult cell cultures fulfilled the usual criteria for characterization as interferon.

Aging↗

PubMed2006

Essential functions of the unique N-terminal region of the varicella-zoster virus glycoprotein E ectodomain in viral replication and in the pathogenesis of skin infection.

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.

Amino Acid Sequence↗

PubMed2005

Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.

Aspirin↗

PubMed1996

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication.

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.

Animals↗

PubMed2006

An increase in viral replicative capacity drives the evolution of protease inhibitor-resistant human immunodeficiency virus type 1 in the absence of drugs.

Little is known about the factors which drive the evolution of protease inhibitor-resistant human immunodeficiency virus type-1 in the absence of drugs. To examine if viral replicative capacity (RC) is an important determinant, we performed in vitro evolution experiments in the absence of drugs with a unique panel of 6 drug-resistant human immunodeficiency virus type-1 recombinant protease variants with a range of different RC. The experiments revealed that an increase in viral RC was indeed an important determinant of evolution. Initial protease inhibitor-resistant viruses with only a few protease mutations and a lowered RC evolved into viruses with an increased RC, either by reversion of primary resistance mutations or by the acquisition of compensatory mutations. For these viruses with a lowered RC, higher fitness peaks are most likely available in the sequence space. Evolution of these viruses in the absence of drugs will therefore drive them to new fitness peaks. In contrast, viruses with an RC comparable to wild type or even higher than wild type did not show any evolution. In the case of these viruses, it is not so likely that higher fitness peaks are present within the sequence space, and therefore, these variants will persist in the absence of drug pressure.

Amino Acid Substitution↗

PubMed1976

Polyoma genome in hamster BHK-21-C13 cells: integration into cellular DNA and induction of the viral replication.

When grown at 39.5 degrees C, BHK-21 C-13 cells transformed by A gene mutants of polyoma virus contain viral sequences that are predominantly associated with cellular DNA pelleted in the Hirt lysis procedure. At this temperature, in cells that are inducible for viral DNA replication (Folk, 1973), the majority of the viral genomes are covalently joined with cellular DNA's containing repetitious sequences. Upon a shift to 31 degrees C, free viral genomes appear and are replicated. Coupled with the replication of the free viral genomes at 31 degrees C is an increase in the viral genomes associated with cellular DNA.

Cell Line↗

PubMed2006

Quantitative detection of hepatitis E virus RNA and dynamics of viral replication in experimental infection.

Hepatitis E virus (HEV) RNA has been detected in the stool and serum of patients with HEV infection and experimentally infected nonhuman primates. However, dynamics of HEV levels in the stool and serum during clinical and subclinical infections have not been determined. A real-time reverse transcription polymerase chain reaction assay, using SYBR Green I in a LightCycler, was developed and optimized to allow quantification of HEV RNA in the stool and serum of both genotype 1 and 2 isolates. The specificity of the assay was confirmed by testing known HEV-RNA-positive and -negative stool and serum specimens and the sensitivity was evaluated using a synthetic HEV RNA standard. Profiles of viraemia and faecal shedding in two chimpanzees inoculated with an isolate of HEV genotype 1 showed the appearance of virus in the stools on day 4 postinoculation (5.65-6.85 log copies/mg) and in the serum on day 7 postinoculation (6.0-6.93 log copies/mL). Peak HEV RNA levels in the stool and serum coincided with peak alanine aminotransferase (ALT) levels observed on day 22 postinoculation in the two chimpanzees. At the time of detection of IgG anti-HEV in serum, viral RNA was no longer detectable in the stool or serum and ALT values had returned to normal levels in both chimpanzees, suggesting the efficacy of the immune response in terminating viral replication. Quantitative evaluation of HEV RNA in humans may allow determining the role of virus levels in the pathogenesis and transmission of HEV.

Animals↗

PubMed1999

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.

Animals↗

PubMed1997

Viral replication in patients with concomitant hepatitis B and C virus infections.

The aim of this study was to assess the implications of dual infection with hepatitis B virus (HBV) and hepatitis C virus (HCV). The HBV and HCV status in 100 patients with chronic hepatitis was analysed. HBV DNA was studied using liquid hybridization and the polymerase chain reaction (PCR). HCV viremia was measured using qualitative and quantitative PCR. The HCV genotype was determined by PCR. Patients were divided into three groups according to their HCV-RNA and HBsAg status: group I consisted of 40 patients with chronic hepatitis caused by HBV; group II, 40 patients with chronic hepatitis caused by HCV; and group III, 20 patients infected with both viruses. The HBV-DNA level was higher in group I than in group III (66.4 vs. 11.5 pg/ml; p < 0.05). Quantification of HCV viremia revealed mean values of 36.9 copies x 10(5)/ml in group II and 5.5 copies/ml x 10(5) in group III (p < 0.05). The mean aminotransferase level and histological activity were higher in group III. HCV genotype lb was the predominant type. The data suggest that there is reciprocal inhibition of viral replication in patients with dual HBV and HCV infection. Liver disease appears to be more severe in patients with chronic hepatitis B and C.

Adolescent↗

PubMed1993

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.

Animals↗