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PubMed2004

Infectious transmission and replication of human T-cell leukemia virus type 1.

Retrovirus infection proceeds by attachment of the envelope glycoprotein to a cell surface receptor, followed by fusion of the viral and cellular membranes. Once in the cell, the viral enzymes and structural proteins form a replication complex that converts the single-stranded viral genomic RNA into a double-stranded DNA, which is then integrated into the host cell chromosome. For HTLV-1, these events are not well characterized. We have developed cell culture systems, infectious molecular clones, and viral vectors that can be used to characterize the mechanisms of HTLV-1 infection and replication. Infection with cell-free HTLV-1 virions is orders of magnitude less efficient compared with other retroviruses. This inefficiency is the result of a block in the replication process after the virion is bound to the cell surface. We are determining whether this block is conferred by the viral replication enzymes, results from the actions of cellular restriction factors, reflects the need for cell-cell contact, or is caused by a combination of these factors.

Animals↗

PubMed1996

Molecular determinants of Theiler's murine encephalomyelitis-induced disease.

Theiler's murine encephalomyelitis virus (TMEV) strains are divided into two subgroups on the basis of their differing disease phenotypes. Members of the GDVII subgroup, such as GDVII strain, produce an acute lethal polioencephalomyelitis. In contrast, members of the TO subgroup, such as DA strain, induce a persistent infection with chronic demyelination; this white matter disease serves as an experimental model of multiple sclerosis (MS) due to their similar pathology and because the immune system in both diseases appears to contribute to the demyelination. The availability of full-length infectious TMEV clones, the relative simplicity of the TMEV genome, and the availability of the mouse as a host provide the opportunity to identify molecular determinants and disease mechanisms that are responsible for neurovirulence, demyelination and virus persistence, and makes this a valuable system for pathogenesis studies.

Animals↗

PubMed2001

Infectivity of wild-type and deleted proviral SIV DNA.

Live attenuated lentiviruses are potentially effective candidate HIV vaccines; however, delivery of these viruses in the field would be problematic. Delivery of attenuated lentiviruses as proviral DNA would be a simple means of immunization, but the efficiency of this method of delivery is not known. In this study, macaques were readily infected following inoculation of plasmid DNA encoding proviral simian immunodeficiency virus (SIVmac239), whether given i.m. (300 microg) or epidermally (15 microg), with all four animals succumbing to AIDS at a mean of 26 weeks following inoculation. Using a human skin explant model, we found that the 50% infectious dose (ID50) of proviral SIV or HIV-1 plasmid may be as low as 1 microg when delivered to skin by gold particle bombardment using a gene gun. An infectious proviral clone of SIV mac239 with a 105 bp deletion in the 3' nef/LTR overlap region was engineered (SIVsbbc delta3), analogous to the initial common nef/LTR deletion in HIV-1 strains isolated from an Australian cohort of long-term slow-progressors. Two further macaques were also readily infected with SIVsbbc delta3 after i.m. injection of 300 microg of highly purified plasmid DNA. Unexpectedly, in one macaque inoculated with SIVsbbc delta3 DNA, SIV strains isolated three to six weeks after infection had completely repaired the nef/LTR deletion with wild-type sequence, and eventually progressed to AIDS. The mechanism used to rebuild this deletion with wild-type sequence, presumably derived from an intact 5' LTR, is unclear, but possibilities include RNA read-through errors from the plasmid DNA and recombination with residual plasmid DNA at the inoculation site.

Animals↗

PubMed2001

Targeted RNA recombination of the membrane and nucleocapsid protein genes between mouse hepatitis virus and bovine coronavirus.

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.

Amino Acid Sequence↗

PubMed1999

Selection and characterization of HIV-1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC).

Human immunodeficiency virus type 1 (HIV-1) variants were selected for resistance against the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC), using the infectious molecular clone HXB2D grown in the MT-4 line of human T cells. The variants selected with (+) dOTC were approximately 6-7-fold less sensitive than wild-type virus to this drug. Cloning and sequencing of the complete reverse transcriptase (RT) coding region of these variants identified the M1841 mutation and further selection with virus containing the M1841 substitution led to the appearance of an M184V mutation. In contrast, selection experiments performed with (-) dOTC yielded variants capable of growing in drug concentrations as high as 100 microM, but phenotypic analysis of these viruses revealed near wild-type 50% inhibitory concentration (IC50) values for this compound. Site-directed mutagenesis experiments in which the M1841 and M184V mutations were introduced into HXB2D confirmed the importance of these mutations when viruses were grown in MT4 cells. However, wild-type IC50 values in regard to both (-) and (+) dOTC were obtained when these recombinant viruses were grown in cord blood mononuclear cells (CBMC). Clinical isolates of HIV-1 resistant to lamivudine and containing the M184V substitution also displayed low-level resistance to both (-) and (+) dOTC when grown in CBMC. Finally, cell-free RT assays were performed in the presence of either (-) dOTC triphosphate, (+) dOTC triphosphate, or the triphosphate of a racemic mixture of (+) and (-) dOTC with wild-type and mutated M184V-containing recombinant RT. The data demonstrate chain termination effects of these compounds with regard to both wild-type and mutated enzyme and that the latter was approximately twofold less sensitive than the former to these drugs.

Anti-HIV Agents↗

PubMed2004

Prostratin induces HIV activation and downregulates HIV receptors in peripheral blood lymphocytes.

Induction of HIV expression through lymphocyte activation has been proposed as a strategy to purge latent reservoirs. Prostratin is a non-tumourogenic phorbol ester that delays HIV replication in vitro, but paradoxically activates HIV expression in latently infected cells. To get a better insight into the mechanisms of action of prostratin, we have analysed the effect of prostratin on HIV activation and HIV receptor and coreceptors' surface expression in human lymphocytes. Peripheral blood mononuclear cells (PBMCs) were transfected with luciferase expression constructs under the control of wild type HIV-long terminal repeat (LTR) and consensus sequences for transcription factors involved in HIV-LTR transactivation (NF-kappaB, SP1, NFAT). Prostratin stimulates transactivation of LTR vectors, kappaB- and SP-1-driven luciferase constructs. In another set of experiments, PBMCs were transfected with a full-length infectious viral clone. Prostratin induced HIV transcription and viral expression as detected by luciferase activity in cellular extracts and p24 levels in culture supernatants, respectively. Expression of the HIV coreceptors CCR5 and CXCR4 was decreased by prostratin and, concomitantly, prostratin inhibited the infection of PBMCs with R5 and X4 strains. However, prostratin did not inhibit infection with a pseudotyped viral clone that enters into the cells independently of HIV receptors. These results help to explain the paradoxical effects of prostratin. On one hand, prostratin induces HIV activation in latently infected cells through the induction of NF-kappaB and Sp1. On the other hand, strong and persistent downregulation of HIV receptors decreases infection of new targets and delays HIV propagation. These data support the potential use of prostratin to activate HIV from latency and purge viral reservoirs.

Cells, Cultured↗

PubMed2004

Genetically modified, live attenuated dengue virus type 3 vaccine candidates.

Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Delta30(ME)) and the absence (rDEN3/4(ME)) of the Delta30 mutation, a previously described 30-nucleotide deletion in the 3' untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Delta30. The chimeric viruses rDEN3/4(ME) and rDEN3/4Delta30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in severe combined immune deficiency mice transplanted with human hepatoma cells, in rhesus monkeys, and in Aedes and Toxorynchites mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4Delta30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3Delta30 was not attenuated in any model tested when compared with wild-type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4Delta30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Amino Acid Sequence↗

PubMed2006

Mutations of conserved glycine residues within the membrane-spanning domain of human immunodeficiency virus type 1 gp41 can inhibit membrane fusion and incorporation of Env onto virions.

The membrane-spanning domain (MSD) of HIV-1 envelope protein (Env) has an additional glycine residue within a well-conserved putative transmembrane helix-helix interaction motif, GXXXG, and forms a G(690)G(691)XXG(694) sequence (G, glycine; X, any residues; the numbering indicates the position within the Env of an infectious molecular clone, HXB2). Different from vesicular stomatitis virus G (VSV-G), the glycine residues of the GXXXG motif of HIV-1 showed higher tolerance against mutations, and a simultaneous substitution of G690 and G694 with leucine residues only modestly decreased fusion activity and replication capacity of HIV-1. When G691 was further substituted with alanine, phenylalanine or leucine residue while G690 and G694 were substituted with leucine residues, the efficiency of membrane fusion decreased, with the decrease greatest occurring with the leucine substitution, a less severe decrease with phenylalanine, and the least severe decrease with alanine. Substitution with leucine residue also decreased the incorporation of Env onto virions, and the mutant showed the most delayed replication profile. Thus the presence of the extra glycine residue, G691, may increase the tolerance of the other two glycine residues against mutations than VSV-G. The fact that a more severe defect was observed for the leucine residue than the phenylalanine residue suggested that the function of Env depended on the steric nature rather than on the simple volume of the side chain of the amino acid residue at position 691. Based on this result, we propose a hypothetical model of the association among MSDs of gp41, in which G(691) locates itself near the helix-helix interface.

Amino Acid Sequence↗

PubMed1990

SIVsmm infection of macaque and mangabey monkeys: correlation between in vivo and in vitro properties of different isolates.

Simian immunodeficiency virus from sooty mangabey monkeys (SIVsmm), a lentivirus closely related to SIV from macaques and the human immunodeficiency virus type 2 (HIV-2), is pathogenic for various species of macaques but is nonpathogenic for mangabeys. Comparison of in vivo and in vitro responses of macaques and mangabeys or their lymphocytes, respectively, to SIVsmm infection indicated that lack of disease in mangabeys apparently was not due to effective control of virus expression by the immune system because SIVsmm-infected, asymptomatic mangabeys have high viral loads. Failure of mangabeys to develop disease may be related to the fact that the prototype SIVsmm (SMM-9) replicated in, but was not cytopathic for, mangabey CD4+ cells. In contrast, replication of SMM-9 in peripheral blood mononuclear cells from pigtailed macaques resulted in specific loss of CD4+ cells and induction of an AIDS-like disease. A variant of SMM-9, designated SMM-PBj14, was identified, however, that was extremely cytopathic for mangabey CD4+ cells and also induced acute lethal disease in both macaques and mangabeys. Acute disease was associated with extensive lymphoid hyperplasia, which was correlated in vitro with induction of proliferation of PBMC in SMM-PBj14-infected cultures. Infectious molecular clones of SMM-PBj14 exhibited the same in vitro and in vivo properties as SMM-PBj14. Future analysis of chimeric viruses may lead to the identification of specific regions of the viral genome that influence the various in vivo and in vitro properties of these SIVsmm isolates.

Animals↗

PubMed1994

Development and evaluation of an HIV-1 transfection-neutralization assay.

We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.

Amino Acid Sequence↗

PubMed1994

Replicating vectors for cancer therapy: a question of strategy.

Direct gene transfer to tumor cells in vivo may have considerable therapeutic potential, provided that a high enough efficiency of gene delivery can be achieved. All other things being equal (e.g. dose, route of administration), a replicating vector should give a higher efficiency of gene transfer than a nonreplicating vector due to local spread from the initially transduced cells. Almost any virus could provide the basis for a replicating gene transfer vector but, for the treatment of human cancer, the choice is greatly constrained by issues of public safety and potential efficacy. Public safety considerations point to viruses of low pathogenicity which are already prevalent in the human population whilst efficacy considerations point to viruses which replicate efficiently in the tumor cells to be targeted. Additional factors influencing the suitability of individual viruses as a basis for replicating vectors include the availability of an infectious molecular clone, the capacity for foreign gene insertion, genomic stability, potential for targeting by coat protein engineering or promoter engineering, cell-cycle dependence and availability of an effective vaccine.

Gene Transfer Techniques↗

PubMed1998

Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir.

The reservoir of human immunodeficiency virus (HIV) in semen is unknown. Several lines of evidence suggest that semen HIV may not arise from the same reservoir of infection as peripheral blood. If true, the viral burden in the two compartments could be qualitatively and quantitatively different, a scenario of potentially profound significance for the design of effective strategies of treatment, disease monitoring, and infection containment. We report here that the ratio of infected to uninfected leukocytes in ejaculated semen specimens is highly discordant with paired blood samples, demonstrating that they derive from distinct populations of infected cells. In addition, infectious HIV was isolated from semen cells, but not from blood cells, of an individual on triple antiretroviral therapy; the absence of major resistance-conferring mutations in the semen virus indicates that it was replicating in isolation from the antiviral agents. The compartmentalization of blood and semen infection was further supported by genetic analysis of several infectious HIV clones isolated from semen cells and peripheral blood cells of another donor not on antiretroviral therapy. Protease gene sequence analyses revealed significant divergence of the two viral populations. These findings confirm the distinct compartmentalization of HIV in the semen of this study cohort, and support the concept that semen HIV arises from an isolated reservoir of infection that may function independently in the pathobiology of HIV disease.

Amino Acid Sequence↗

PubMed1998

[Studies on bovine leukemia virus envelope glycoprotein gp30 YXXL sequences in virus infection and fusogenic activity].

The bovine leukemia virus (BLV) envelope transmembrane protein (gp30) contains three YXXL sequences at its cytoplasmic tail. It is known that N-terminal two of these sequences participate in the induction of B cell activation when chimeric proteins in which cytoplasmic domain of CD8-alpha has been replaced with that of BLV gp30 are stimulated by anti-CD8-alpha antibody. In addition to such signal transduction activity, the two tyrosines in the YXXL sequences also appear to involve infection with high viral loads and their maintenance in the sheep experimentally infected with BLV. To analyze detailed biological relevance of these sequences in vitro, we constructed a full-length BLV-infectious molecular clone with two copies of long terminal repeats (LTRs), designated pBLV-IF, and then changed residues Y487, L490, Y498, L501 or Y487 plus Y498 in gp30 cytoplasmic tail to Ala by site-directed mutagenesis. Introduction of molecular clones of wild-type and mutants into COS-1 cells revealed that all mutated molecular clones synthesized matured envelope proteins and released virus particles into growth medium. Serial passages of transient transfectants with the molecular clones and cell-free inoculation resulted in the reduction of infectivity by mutation of Y498 and Y487 plus Y498. In viral penetration, but not specific virus-cell binding, mutations of Y498 and Y487 plus Y498 substantially reduced the potential. Mutations of Y487, Y498 and Y487 plus Y498 increased syncytium-forming potential with increasing expression of envelope proteins on cell surface. In contrast, mutations of L490 and L501 affected neither infectivity nor syncytium-formation. Altogether, our data indicate that the YXXL sequences play a critical role during virus penetration and fusogenesity in the life cycle of BLV and regulate surface expression of envelope proteins.

Amino Acid Sequence↗

PubMed1989

Complete nucleotide sequence of a molecular clone of woodchuck hepatitis virus that is infectious in the natural host.

Woodchuck hepatitis virus (WHV) DNA was cloned from viral particles obtained from the serum of a woodchuck with a naturally acquired infection. The complete nucleotide sequence of the virus genome was determined and found to be 3323 base pairs long. Transfection experiments demonstrated that the recombinant WHV DNA was infectious in each of 18 woodchucks tested and established a chronic carrier state in 1 of 13 neonates and 3 of 5 adult animals. WHV DNA from serum particles from the chronically infected neonate was cloned and the nucleotide sequence of three independent recombinants was compared directly with that of the input recombinant DNA. The consensus sequence of the three progeny genomes was identical to that of the parental DNA sequence. Therefore, transfection of woodchuck livers with recombinant WHV DNA induces active virus replication and gene expression and yields progeny genomes that are faithful copies of the input virus genome.

Animals↗

PubMed1996

Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y.

Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7 kb PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immuno-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.

Cloning, Molecular↗

PubMed1983

Construction of infectious potato spindle tuber viroid cDNA clones.

Contiguous restriction fragments from two cloned partial-length potato spindle tuber viroid (PSTV) cDNAs were used to construct recombinant DNAs containing full-length monomeric and dimeric PSTV cDNA. When five different PSTV cDNA plasmids and RNA isolated from E. coli cells harboring these plasmids were tested for infectivity on tomato, plasmid DNAs containing PSTV cDNA dimers were infectious. RNA transcripts containing the sequence of PSTV from these plasmids were also infectious. The sequences of the viroid progeny and the cloned DNA were identical. In vitro mutagenesis of infectious PSTV cDNAs will allow systematic investigation of the role of specific sequences in viroid replication and pathogenesis.

Base Sequence↗

PubMed1991

Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus.

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I-mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.

Animals↗

PubMed1995

Assessing genetic-based therapies for AIDS using the simian immunodeficiency virus.

A plasmid encoding the full-length infectious molecular proviral clone of SIVmac239 was generated. Virus derived from cells transfected with this clone replicated to high levels and was cytopathic for some transformed human CD4+ cell lines and primary rhesus macaque peripheral blood mononuclear cells. Since replication of SIV requires the functional expression of the viral encoded rev protein, transient co-transfection studies were initiated with the infectious proviral clone and a well-characterized trans-dominant negative HIV-1 rev mutant.

Animals↗