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PubMed2004

Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity.

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.

Animals↗

PubMed1998

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction.

The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models.

Animals↗

PubMedDate not supplied

Cucumber mosaic virus from Ixora: infectious RNA transcripts confirm deficiency in satellite support and unusual symptomatology.

A cucumber mosaic virus isolate from an Ixora plant originating in the Philippines and first described 20 years ago (CMV-Ix) by Waterworth and Povish differs from other characterized CMV strains in its anomalous satellite support and in several biological and molecular properties. We describe the preparation of infectious transcripts from cloned complementary DNA and the characterization of progeny virus. The results confirm that CMV-Ix has a more limited host range than most CMV isolates. Virions of CMV-Ix are smaller than those of the control CMV-S, independent of the type of negative stain used. Furthermore, CMV-Ix from transcripts supports the replication of the satellite T-CARNA 5 but not of D-CARNA 5.

Cloning, Molecular↗

PubMed1980

Sequence of retrovirus provirus resembles that of bacterial transposable elements.

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified lambda phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

Base Sequence↗

PubMed2002

Advances in hepatitis C: what is coming in the next 5 years?

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Numerous advances have been made in the understanding of HCV replication, including detailed molecular characterization of its viral proteins and genomic RNA. The inability to grow HCV in cell culture had impeded the development of antiviral agents against this virus. To overcome this obstacle, a number of unique tools have been prepared, such as molecular clones that are infectious in the chimpanzee animal model of infection, and the development of a subgenomic replicon system in Huh7 cells. In addition, the major non-structural proteins have been crystallized, thus enabling rational drug design directed to these targets. Current developments in antiviral agents are reviewed in the context of these potential new viral targets for the future treatment of HCV in chronically infected individuals.

Antiviral Agents↗

PubMed1976

Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.

Cell Line↗

PubMed1982

Spontaneous changes in nucleotide sequence in proviruses of spleen necrosis virus, an avian retrovirus.

We determined the nucleotide sequence of about 1 kilobase of DNA 3' to the 5' long terminal repeat of three noninfectious ad one infectious proviral DNA clones of spleen necrosis virus, an avian retrovirus, to determine if the types of nucleic acid changes involved in retrovirus mutation shed light on special features of retrovirus replication. An open reading frame was found starting 411 base pairs from the end of the long terminal repeat. It contained sequences coding for the 36 amino acids at the amino terminus of the p30 of a related reticuloendotheliosis virus [Oroszlan, S., Barbacid, M., Copeland T., Aaronson, S. A. & Gilden, R. V. (1981) J. Virol. 39, 845-854]. Therefore, the open reading frame represents the 5' end of the gag gene. A mutation in one noninfectious provirus changed the initiation codon for the gag polypeptide; a mutation in another noninfectious provirus caused premature termination of gag polypeptide synthesis; and a nontandem duplication into gag resulting from a mistake in initial (+) strand DNA synthesis changed amino acids and the reading frame in a third noninfectious provirus. These mutations appear to be responsible for the lack of infectivity of these provirus clones and indicate a higher relative frequency of mutation in this region of the genome. In addition, all four clones have multiple other mutations. These mutations are mostly base pair substitutions and many are clustered for any one clone, reflecting certain special features of retrovirus replication.

Amino Acid Sequence↗

PubMed1986

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome.

We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature.

Base Sequence↗

PubMed1991

Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein.

It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptom-attenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general.

Base Sequence↗

PubMed1991

Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection.

Alterations in two highly conserved N-linked glycosylation sites within the gp120 envelope glycoprotein of human immunodeficiency virus type I (HIV-1) implicated in the phenotype of a noncytopathic HIV-1 variant were introduced independently and in combination into a cytopathic, infectious HIV-1 clone by site-specific mutagenesis. Neither mutation affected the synthesis of HIV-1 envelope glycoproteins. However, one of the mutations restricted the ability of HIV-1 envelope to localize on the cell membrane and thus markedly impaired virus assembly. The HIV-1 assembly defect could be overcome in trans if site-specific mutants were packaged in HeLa cells constitutively producing wild-type HIV-1 envelope glycoprotein. In addition to inefficient virus assembly, this mutation impaired the ability of the virus to infect CD4+ T cells, but did not affect CD4-independent infection of muscle cells. These results suggest additional functions of posttranslational modification in virus replication (i.e., envelope glycoprotein transport). Given that such modifications can restrict CD4-mediated uptake without affecting CD4-independent uptake, variations in posttranslational env processing between different HIV-1 genotypes may affect virus tropism in vivo.

Amino Acid Sequence↗

PubMed1994

SIVmac expressing hybrid envelope proteins containing HIV-1 V3 and/or C4 sequences is not competent for replication.

The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone were replaced by the corresponding HIV-1 sequences. Viral particles were obtained after transfection of COS-1 cells. Chimeric SIVmac constructs were not replication competent in the human T cell lines CEMx174, AA2, H9, and MT-4 or in primary cultures of rhesus monkey peripheral blood mononuclear cells. The lack of infectivity of the hybrid constructs was associated with inefficient proteolytic processing of the gp160env precursor. Unlike the modular nature of some proteins, gp120 appears to be a highly ordered molecule whose function is dependent on the integration of many discontinuous, interactive regions.

Amino Acid Sequence↗

PubMed1995

Enhanced susceptibility to human immunodeficiency virus infection in CD4+ T lymphocytes genetically deficient in CD43.

CD43 is a cell surface sialoglycoprotein expressed by most cells of hematopoietic origin, including all T lymphocytes. Elimination of CD43 expression by gene targeting in the CEM T cell line results in its increased homotypic adhesion and binding to HIV-1 gp120. Here we report that the CD43-negative CEM cells show increased susceptibility to HIV-1 infection and increased viral replication compared with the parental CD43+ CEM cell line. Increased HIV-1 replication also was observed in CEM cells with diminished CD43 expression secondary to functional inactivation of a single CD43 allele. The CD43- CEM cells were more susceptible to HIV-1-induced cytopathicity than their CD43+ counterparts. HIV-1 replication also was increased in the CD43- CEM cells after transfection with the infectious HIV molecular clone pNL4-3. These data suggest that factors that diminish CD43 expression on T lymphocytes may enhance their susceptibility to HIV-1 infection.

Antigens, CD↗

PubMed1995

Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function.

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.

Animals↗

PubMed1998

On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.

Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We introduced a stop codon at the end of the first Tat-coding exon in an expression vector that produces a truncated 71-amino acid Tat protein. This Q72stop mutant displays reduced transcriptional activity of approximately 54% in transient LTR-CAT transfection assays. To test the contribution of the second Tat-coding exon to virus replication, the Q72stop mutation was also introduced in the infectious pLAI molecular clone. The effect on virus replication was analyzed in primary cells and in a transformed T cell line. The fitness of the mutant virus was calculated to be approximately 75% compared with the wild-type control. Thus, a small contribution of the C-terminal Tat domain to viral fitness was measured. It has been proposed that the second Tat-coding exon is involved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was analyzed in T cells infected with the wild-type LAI virus and the replication-competent Q72stop mutant. MHC expression was transiently reduced on infection with either virus, indicating that the second Tat-coding exon is not involved in this downregulation.

Animals↗

PubMed1988

Transfer and expression of the bacterial NPT-II gene in chick embryos using a Schmidt-Ruppin retrovirus vector.

In an effort to introduce foreign genes into chickens, the bacterial neomycin phosphotransferase (NPT-II) gene was cloned into an infectious avian retroviral vector derived from the Schmidt-Ruppin A strain of RSV. The NPT-II gene was stable in the vector during passage in vitro and infected cells were resistant to G418. Fertilized chicken embryos were inoculated with the recombinant virus on day 0 and screened on day 20 for the NPT-II gene in blood cell DNA. Approximately 12% of the embryos were positive for the NPT-II gene. Screening of DNA from the brain, muscle, liver and foot of the positive embryos indicated that the NPT-II gene copy number could vary in a single embryo. However, some embryos had nearly equal NPT-II copy number in each tissue examined. To determine the expression of the bacterial gene, tissue extracts from the positive embryos were assayed for NPT-II activity. The results indicated that NPT-II activity varied depending on the tissue, with activity being highest in muscle and foot regardless of NPT-II gene copy number.

Animals↗

PubMed1996

Myopathy and spontaneous Pasteurella pneumotropica-induced abscess formation in an HIV-1 transgenic mouse model.

In an effort to augment human immunodeficiency virus type 1 (HIV-1) gene expression in transgenic mice, an infectious proviral DNA clone was modified by deleting the two NF kappa B binding sites and some adjacent upstream LTR sequences and replacing them with the core enhancer of Moloney murine leukemia virus (MLV). Two independent lines of MLV/HIV transgenic mice were established that expressed HIV-1-specific RNA in lymphoid tissue, striated skeletal muscle, and the eye lens. Heterozygous animals from each transgenic line spontaneously developed an inflammatory disease of the eye associated with the production of copious amounts of purulent lacrimal secretions beginning at 2 weeks of age. Periorbital abscess formation became grossly apparent by 2 months of age and Pasteurella pneumotropica was cultured from the harderian glands and conjunctival surfaces of many of the MLV/HIV animals but not their nontransgenic, cohabiting littermates. This gram-negative commensal bacterium has been previously associated with a similar disease phenotype in immunocompromised (e.g., nude mice) rodent colonies. MLV/HIV mice developed normally until 15 weeks of age, when weight loss and wasting occurred, culminating in premature death (as earlier as 6 months of age). The cachexia was associated with an initially focal and subsequently progressive myopathy, coinciding with age-related increases of HIV gene expression in muscle.

Abscess↗

PubMed1993

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

Base Sequence↗

PubMed1997

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Base Sequence↗