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PubMed1989

Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.

Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters. The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail. Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA. Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG). Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.

Blotting, Western↗

PubMed1989

Nucleotide sequence and genomic organization of feline immunodeficiency virus.

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.

Amino Acid Sequence↗

PubMed1991

The transcriptional transactivator of human foamy virus maps to the bel 1 genomic region.

The human foamy virus (HFV) genome possesses three open reading frames (bel 1, 2, and 3) located between env and the 3' long terminal repeat. By analogy to other human retroviruses this region was selected as the most likely candidate to encode the viral transactivator. Results presented here confirmed this and showed further that a deletion introduced only into the bel 1 open reading frame of a plasmid derived from an infectious molecular clone of HFV abolished transactivation. In contrast, deletions in bel 2 and bel 3 had only minor effects on the ability to transactivate. The role of the bel 1 genomic region as a transactivator was further investigated by eukaryotic expression of a genome fragment of HFV spanning the bel 1 open reading frame. A construct expressing bel 1 under control of a heterologous promoter was found to transactivate the HFV long terminal repeat in a dose-dependent fashion. Furthermore, it is shown that the U3 region of the HFV long terminal repeat is sufficient to respond to the HFV transactivator.

Animals↗

PubMed1993

Mapping the determinants of human immunodeficiency virus 2 for infectivity, replication efficiency, and cytopathicity.

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Down-regulation of Rous sarcoma virus long terminal repeat promoter activity by a HeLa cell basic protein.

We have previously isolated a HeLa cell cDNA encoding a 21-kDa polypeptide that is 48% similar to transcription factor IIS. To explore the possibility that p21 plays a role in transcriptional regulation in vivo, we tested the effect of p21 expression on the synthesis of reporter chloramphenicol acetyltransferase (CAT) in transfected COS-1 cells. CAT formation under control of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter was decreased nearly 20-fold in cells coexpressing p21. In contrast, CAT production under control of other sequence elements was only slightly reduced (human immunodeficiency virus type 1 LTR, simian virus 40 early promoter), unaffected (human heat shock protein of 70-kDa promoter, adenovirus major late promoter TATA box), or increased (terminal deoxynucleotidyltransferase initiator element, c-fos promoter) by p21 coexpression as compared to cells cotransfected with the parental vector. The abundance of steady-state CAT transcripts from RSV LTR was also decreased by p21 expression in a dose-dependent manner, suggesting that transcription of RSV LTR/CAT is under negative control by p21. Consistent with an effect on transcription, p21 was localized in nuclei of transfected cells. Deletion analysis of p21 indicated that the sequences essential for inhibition of RSV LTR function include the previously identified ARg/Ser-rich region and zinc finger-like motif. Proliferation of chicken embryo fibroblasts transfected with an infectious molecular clone of RSV was diminished by p21 expression, which also resulted in fewer transformed foci.

Animals↗

PubMed1996

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

Antiviral Agents↗

PubMed1996

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Base Sequence↗

PubMed1999

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences.

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.

5' Untranslated Regions↗

PubMed2001

Maintenance of glycoprotein-determined phenotype in an HIV type 1 (pNL43) env gene-cassetting system.

Here we report the construction and use of a full-length env gene-cassetting system, C2, based on the HIV-1 infectious molecular clone NL43. C2 produces virus with the same phenotype as NL43 but with 2-fold lower growth kinetics. The latter probably relates to alteration in the vpu and/or nef genes. C2-env chimeras of macrophage-tropic and T cell-tropic laboratory strains and primary HIV-1 isolates retain the glycoprotein-determined phenotypes of their parent viruses. The cassette will assist studies of HIV-1 pathogenesis.

Amino Acid Sequence↗

PubMed1992

Differential tropism of clinical HIV-1 isolates for primary monocytes and promonocytic cell lines.

Previously we demonstrated a correlation between a nonsyncytium-inducing (NSI), non-T-cell line tropic phenotype of HIV-1 isolates and the capacity to replicate in primary monocyte-derived macrophages (MDM). Here we demonstrate that these NSI, monocytotropic HIV-1 isolates lack the capacity to replicate in two promonocytic cell-lines, HL60 and U937. In contrast, most syncytium-inducing (SI) HIV-1 isolates with tropism for T-cell lines and generally non-monocytotropic were able to establish a productive infection in promonocytic cell lines. Similar differences in tropism for monocytes and promonocytic cell lines were observed with infectious molecular clones. Our results indicate that virological studies on promonocytic cell lines do not necessarily pertain to the HIV-1 infection of monocytes in vivo.

Cell Line↗

PubMed1994

Immunogenicity of Nef protein of SIVSMM-PBj14 expressed in a live vaccine strain of Salmonella species.

The nef gene of an infectious molecular clone of SIVSMM isolate PBj14 was fused to the glutathione S-transferase gene of Schistosoma japonicum to generate plasmid pEMC100. The recombinant plasmid was placed in an aroA live vaccine Salmonella dublin strain, and the production of GST-Nef protein was induced by exposure to IPTG. The fusion protein was purified and administered as vaccine to BALB/c mice by i.p. injection. Several doses of the purified fusion protein produced an earlier anti-GST-Nef response, without an anti-GST response, than did IPTG-induced Salmonella live vaccine containing an equal amount (0.1 microgram) of fusion protein, apparently because of the transient immunosuppressive effect of live vaccine given by injection. The highest anti-GST-Nef titers were obtained by a third immunization schedule in which mice were treated with a priming inoculum of induced live vaccine followed, after the predicted immunosuppressed interval, by two i.p. doses of 1 microgram of purified GST-Nef protein with Ribi adjuvant. The data presented here demonstrate that SL5928 aroA, an attenuated S. dublin strain, can be used as a live vaccine carrier to express Nef protein of SIVSMM-PBj14, one of the most acutely pathogenic primate lentiviruses so far described.

Adjuvants, Immunologic↗

PubMed1994

Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes.

Auxiliary genes that are not essential for viral replication in cell culture are found in all known lentiviruses. The "nonessential" auxiliary genes of HIV-1 are vif, vpr, vpu, and nef. Sequences within the upstream region of U3 in the LTR are also not required for virus replication in cell culture. We constructed a panel of 23 mutants with single and combination deletions in these regions in the wild-type HIV-1 infectious molecular clone NL4-3. Deletion of the vpu, vpr, and nef genes and the U3 upstream sequence (US), individually or in combinations, did not appreciably alter virus replication in either chimpanzee PBMCs, human PBMCs, or in the B/T cell hybrid line CEMx174. In contrast, deletion of the vif gene dramatically delayed virus replication in all three cell types. This collection of HIV-1 deletion mutants will be useful for elucidating the functions of these genes and for investigating antiviral immunity in animal models.

Animals↗

PubMed1995

Humoral and cellular immune responses in rhesus macaques infected with human immunodeficiency virus type 2.

Eighteen rhesus macaques were inoculated with either an infectious molecularly cloned human immunodeficiency virus type 2 (HIV-2)SBL/ISY, or with one of eight mutants defective in one or more accessory genes. The immune responses generated by the macaques were monitored for up to 2 years postinfection. All the macaques except those that received mutants lacking the vpr or vif genes demonstrated low to moderate antibody titers. Macaques inoculated with vpx- mutants exhibited a persistent serological response, suggesting continuous virus expression even in the absence of detectable virus in the peripheral blood mononuclear cells (PBMCs). Neutralizing antibodies developed in only four macaques. In general, low-level cytotoxic T lymphocyte (CTL) activity, not clearly HIV-2 specific, was detected in PBMCs. However, one virus-negative macaque exhibited significant HIV-2-specific CTL activity in an enriched CD8+ cell population from PBMCs, suggesting clearance of the viral infection. In addition, CTL activity against the Env and Gag/Pol epitopes of HIV-2 by CD8+ lymphocytes from the spleens and lymph nodes of two infected macaques, in one case requiring CD8+ T cell enrichment and in the other clearly evident in unfractionated tissue lymphocytes, was demonstrated for the first time. This sequestration of tissue CTLs occurred in the absence of significant levels of circulating CTLs in the blood. Our results suggest that routine monitoring of PBMCs may sometimes be inadequate for detecting cell-mediated immune responses. Elucidation of immune correlates of vaccine protection may therefore require sampling of lymphoid tissues and assessment of enriched CD8+ populations.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.

Base Sequence↗

PubMed1996

Structure-infectivity analysis of the human rhinovirus genomic RNA 3' non-coding region.

The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary structure probing techniques, we have demonstrated the existence of a single stem-loop structure contained entirely within the 3' NCR, which appears to be phylogenetically conserved within the rhinovirus genus. We also report the in vivo analysis of a number of 3' NCR deletion mutations engineered into infectious cDNA clones which were designed to disrupt the stem-loop secondary structure to varying degrees. Large deletions (up to 37 nt) resulted in defective growth phenotypes, although they were not lethal. We propose that the absolute requirements for initiation of negative strand synthesis are less stringent than previously postulated, even though defined RNA secondary structure determinants may have evolved to facilitate and/or regulate the process of viral RNA replication.

Base Sequence↗

PubMed2002

Down regulation of human immunodeficiency virus type-1 (HIV-1) expression by Vif antisense RNA expression vectors in transfected cells.

The HIV-1 vif gene is a potential candidate in the quest for anti-retroviral interventions, due to its unique role in the target cell infection. We employed the antisense RNA strategy to determine the antiviral activity of intracellularly expressed anti-sense RNAs of various lengths complementary to the targeted HIV-1 vif gene. Expression vectors mediating the delivery of the vif-ORF, 5'-Vif, M-vif, and 3'-vif antisense RNAs under the CMV promoter were constructed using pcDNA 3.1. The COS cells transfected with the antisense vectors showed a steady-state of antisense RNA expression levels. In contrast, those co-transfected with the Infectious molecular clone, pNL-E, exhibited a significant reduction in the steady-state antisense RNA levels, which correlated with a significant reduction in p24 antigen production. Thus, this expression method for these antisense RNAs provides a promising gene therapy strategy for HIV-1.

Animals↗

PubMed2000

The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

Brassica↗

PubMed2001

Lettuce mosaic virus pathogenicity determinants in susceptible and tolerant lettuce cultivars map to different regions of the viral genome.

Full-length infectious cDNA clones were constructed for two isolates (LMV-0 and LMV-E) of Lettuce mosaic virus (LMV), a member of the genus Potyvirus. These two isolates differ in pathogenicity in susceptible and tolerant-resistant lettuce cultivars. In susceptible plants, LMV-0 induces mild mosaic symptoms, whereas LMV-E induces severe stunting, leaf deformation, and a necrotic mosaic. In plants carrying either of the two probably allelic recessive resistance genes mol1 or mol2, LMV-0 is restricted partially to the inoculated leaves. When a systemic invasion does occur, however, symptoms fail to develop. LMV-E overcomes the protection afforded by the resistance genes, resulting in systemic mosaic symptoms. Analysis of the behavior of recombinants constructed between the two virus isolates determined that the HC-Pro protein of LMV-E causes the severe stunting and necrotic mosaic induced by this isolate in susceptible cultivars. In contrast, the ability to overcome mol resistance and induce symptoms in the resistant-tolerant cultivars was mapped to the 3' half of the LMV-E genome. These results indicate that the ability to induce severe symptoms and to overcome the protection afforded by the recessive genes mol1 or mol2 are independent phenomena.

Cloning, Molecular↗