Tenofovir treatment in patients with lamivudine-resistant hepatitis B mutants strongly affects viral replication.
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Adenine↗
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Results for “viral replication”
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The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.
The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.
Amino Acid Sequence↗
Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes.
Intracellular expression of antisense transcripts was evaluated for its potential to interfere with human immunodeficiency virus type 1 (HIV-1) replication. Retroviral vectors encoding HIV-1 psi-gag complementary sequences downstream of a selectable gene (neo, puromycin gene, or Lyt2 gene) were stable and yielded high titers. Human CEMSS T cells were transduced with amphotropic retroviral vectors to express RNA complementary to the psi-gag sequence of HIV-1. Replication of laboratory-adapted HIV-1 strains was inhibited by more than 1 order of magnitude (log10) in these transduced cells even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses). Antisense-mediated anti-HIV efficacy was further demonstrated by survival of CD4+ cells in these cultures relative to controls. The level of anti-HIV-1 activity of the psi-gag antisense sequence correlated with the length of the antisense transcript. Maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication. Expression of psi-gag antisense RNA also reduced HIV-1 JR-CSF replication 10-fold in primary CD4+ lymphocytes. These results obtained with a T-cell line and primary peripheral blood lymphocytes indicate the potential of long antisense RNAs as an efficient anti-HIV-1 therapeutic agent for gene therapy.
Anti-HIV Agents↗
[HBV-related proteins as a marker of viral replication].
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Biomarkers↗
Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication.
The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.
Adaptor Proteins, Signal Transducing↗
Antibody-targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication.
Mouse L929 cells were incubated with antibody-targeted liposomes containing oligodeoxyribonucleotides (oligomers). When the oligomer was a 15-mer complementary to the 5'-end region of the mRNA encoding the N protein of vesicular stomatitis virus, the cells became less permissive for multiplication of that virus; greater than 95% reduction of viral multiplication was achieved. Protection was not seen for "empty" liposomes, liposomes containing a random oligomer sequence, or liposomes containing a sequence complementary to the 5' end of c-myc protooncogene mRNA targeted by the same antibody, nor was it seen when the liposomes containing the N-protein antisense oligomer were targeted by an antibody that does not bind to L929 cells. Antibody-bearing liposomes containing antisense oligomers thus have a double specificity: a particular cell selected by the targeting antibody on the liposome and a particular mRNA in the cell selected by sequence complementarity with the liposome-encapsulated oligomer. Nonencapsulated oligomers are sensitive to nucleases and usually must be administered to cells at high concentrations. Oligomers encapsulated in liposomes resist DNase and are active in amounts 1-2 orders of magnitude lower than for those reported for unencapsulated oligomer sequences.
1,2-Dipalmitoylphosphatidylcholine↗
The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.
The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C. Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein beta-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 x g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.
Amino Acid Sequence↗
Role of the nuclear matrix during viral replication.
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Animals↗
Molecular biology of Porcine circovirus: analyses of gene expression and viral replication.
The rep gene of Porcine circovirus type 1 directs the synthesis of two proteins. The full-length protein Rep is 312 amino acids in size, the spliced variant Rep' is truncated (168 aa) and exon 2 is frame-shifted. Replication of PCV1 DNA depends on synthesis of both proteins. Rep and Rep' bind in vitro to double-stranded DNA fragments comprising part of the origin of replication of PCV1, but the minimal binding sites of the two proteins are distinct. Rep protein represses the promoter of the rep gene by binding to the two inner hexamers H1 and H2. Although Rep' binds to the same sequence, it does not influence Prep. Twelve hours after PCV1 infection, similar amounts of rep and rep' were detected by real-time PCR, but later on, the ratio of the two transcripts varied. Both proteins are co-localised in the nucleus and formation of homo- and heteromeric complexes has been observed. When a replication assay was performed, in which Rep and Rep' protein of PCV1 was used to replicate the origin of PCV1 and PCV2, the rep gene products were found to initiate replication at both origins of replication.
Amino Acid Sequence↗
The latency-associated nuclear antigen of rhesus monkey rhadinovirus inhibits viral replication through repression of Orf50/Rta transcriptional activation.
Rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8. We have previously reported that the transcript for RRV latency-associated nuclear antigen (R-LANA) is expressed during lytic replication in rhesus fibroblasts. In this article, we report the development of a latent culture system for RRV and show that mRNA specific for R-LANA is expressed during latency as well. We have characterized the R-LANA protein and demonstrate that it exhibits a nuclear speckled localization and possesses the ability to homodimerize. When expressed in rhesus fibroblasts, R-LANA can inhibit RRV lytic replication in vitro. We have investigated the mechanism behind this inhibition and find that, while R-LANA itself has very little effect on lytic promoters, it can dramatically decrease the transactivation function of RRV Orf50 (Rta), which is the major viral transcription factor. We further show that the mechanism for this repression involves the recruitment of histone deacetylase complexes (HDACs), because R-LANA's ability to repress Orf50 transactivation is completely reversed by the addition of the HDAC inhibitor trichostatin A (TSA). We also report that TSA alone can significantly reactivate RRV from latently infected cells. We propose that the repressive effects of R-LANA on RRV Orf50 transactivation serve to downregulate the transcription of early genes at late times during the lytic cycle and also help to maintain viral latency by preventing viral reactivation.
Animals↗
High-dose nevirapine in previously untreated human immunodeficiency virus type 1-infected persons does not result in sustained suppression of viral replication.
High-dose nevirapine treatment has been reported to confer sustained antiretroviral effects, despite a rapid development of resistance. The use of this strategy was evaluated in 20 previously untreated human immunodeficiency virus type 1 (HIV-1) p24 antigenemic persons with CD4 cell counts between 100 and 500/mm3. Treatment consisted of 400 mg of nevirapine, after a 2-week lead-in dose of 200 mg. Rash was the most frequently reported adverse event, occurring in 25%. While sustained declines in p24 antigen levels were observed in the majority, serum HIV-1 RNA load and CD4 cell counts returned to baseline values within 12 weeks in virtually all subjects. The resistance-conferring tyrosine-to-cysteine substitution at reverse transcriptase position 181 was detected after 4 weeks in most subjects. These observations suggest that plasma drug levels attained with high-dose nevirapine were not sufficient to inhibit nevirapine-resistant virus, although they were approximately 2-fold higher than reported IC50 values of resistant virus.
Acquired Immunodeficiency Syndrome↗
The 2.2-kilobase latency-associated transcript of herpes simplex virus type 2 does not modulate viral replication, reactivation, or establishment of latency in transgenic mice.
To better understand the mechanisms responsible for the observed effects of deletions in the promoter region of the latency-associated transcript (LAT) gene in impairing herpes simplex virus (HSV) reactivation, we generated mice transgenic for a 5.5-kb HSV type 2 (HSV-2) genomic fragment spanning the major LAT, along with the LAT promoter and flanking regions, in the C57BL/6 background. The mice expressed abundant 2.2-kb major LATs in trigeminal ganglia (TG) and other tissues. The effects of the transgene on HSV-2 infection, latency, and reactivation were assessed. When infected with wild-type (WT) HSV-2 or its LAT promoter deletion (LAT(-)) mutant, primary lung fibroblast lines established from normal C57BL/6 and transgenic mice supported virus growth equally well. The replication of these viruses in the mouse eye and their spread to TG and brains were similar. The quantities of latent viral DNA in TG of transgenic and normal mice, as determined by real-time PCR, were comparable. UV light-induced reactivation of the LAT(-) mutant from transgenic mice (0 to 7%) was no more frequent than that from normal mice (0 to 14%), while WT virus was reactivated from 13 to 54% of normal mice and 22 to 54% of transgenic mice. The cumulative data indicate that, when expressed transgenically, the HSV-2 major LAT cannot influence HSV-2 infection or latency and cannot complement the defect in reactivation of the LAT(-) mutant. These results imply that the phenotype of reduced reactivation associated with the LAT(-) mutant is related to a function encoded in the LAT promoter but not to the major LAT itself.
Animals↗
Importance of sequence and structural elements within a viral replication repressor.
Efficient replication of plus-strand RNA viruses requires a 3' proximal core promoter and an increasingly diverse inventory of supporting elements such as enhancers, repressors, and 5' terminal sequences. While core promoters have been well characterized, much less is known about structure-functional relationships of these supporting elements. Members of the genus Carmovirus family Tombusviridae contain a hairpin (H5) proximal to the core promoter that functions as a repressor of minus-strand synthesis in vitro through an interaction between its large symmetrical internal loop (LSL) and 3' terminal bases. Turnip crinkle virus satellite RNA satC with the H5 of carmovirus Japanese iris necrosis virus or Cardamine chlorotic fleck virus (CCFV) did not accumulate to detectable levels even though 3' end base-pairing would be maintained. Replacement of portions of the satC H5 with analogous portions from CCFV revealed that the cognate LSL and lower stem were of greater importance for satC accumulation than the upper stem. In vivo selex of the H5 upper stem and terminal GNRA tetraloop revealed considerable plasticity in the upper stem, including the presence of three- to six-base terminal loops, allowed for H5 function. In vivo selex of the lower stem revealed that both a stable stem and specific base pairs contributed to satC fitness. Surprisingly, mutations in H5 had a disproportionate effect on plus-strand accumulation that was unrelated to the stability of the mutant plus-strands. In addition, fitness to accumulate in plants did not always correlate with enhanced ability to accumulate in protoplasts, suggesting that H5 may be multifunctional.
Base Sequence↗
The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo.
The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.
Base Sequence↗
Role of primary constitutive phosphorylation of Sendai virus P and V proteins in viral replication and pathogenesis.
Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.
Animals↗
Immunoreceptor tyrosine-based activation motif-dependent signaling by Kaposi's sarcoma-associated herpesvirus K1 protein: effects on lytic viral replication.
The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 gene encodes a polypeptide bearing an immunoreceptor tyrosine-based activation motif (ITAM) that is constitutively active for ITAM-based signal transduction. Although ectopic overexpression of K1 in cultured fibroblasts can lead to growth transformation, in vivo this gene is primarily expressed in lymphoid cells undergoing lytic infection. Here we have examined function of K1 in the setting of lytic replication, through the study of K1 mutants lacking functional ITAMs. Expression of such mutants in BJAB cells cotransfected with wild-type K1 results in dramatic inhibition of K1 signal transduction, as judged by impaired activation of Syk kinase and phospholipase C-gamma2 as well as by diminished expression of a luciferase reporter gene dependent upon K1-induced calcium and Ras signaling. Thus, the mutants behave as dominantly acting inhibitors of K1 function. To assess the role of K1 in lytic replication, we introduced these K1 mutants into BCBL-1 cells, a B-cell lymphoma line latently infected with KSHV, and induced lytic replication by ectopic expression of the KSHV ORF50 transactivator. Expression of lytic cycle genes was diminished up to 80% in the presence of a K1 dominant negative mutant. These inhibitory effects could be overridden by tetradecanoyl phorbol acetate treatment, indicating that inhibition was not due to irreversible cell injury and suggesting that other signaling events could bypass the block. We conclude that ITAM-dependent signaling by K1 is not absolutely required for lytic reactivation but functions to modestly augment lytic replication in B cells, the natural reservoir of KSHV.
B-Lymphocytes↗
Effect of suramin on serum viral replication in feline leukemia virus-infected pet cats.
Suramin treatments were administered (IV) to 2 healthy adult cats infected with naturally acquired feline leukemia virus. Serum viral infectivity--as assessed by focus induction by the method of Fischinger, using serial serum samples titrated on clone 81 cells--ceased transiently in both cats during treatment with suramin, but returned to significantly high levels approximately 14 days after treatment was stopped. Both cats tested positive for FeLV internal antigens in peripheral blood cells and serum before, during, and after treatment. Both cats tested negative for antibody to feline oncornavirus membrane antigen before, during, and after treatment. The major adverse effects of suramin in the 2 cats were transient vomiting and anorexia.
Animals↗
SV40-adenovirus "hybrids": presence of two genotypes and the requirement of their complementation for viral replication.
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Adenoviridae↗