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PubMed1987

Complete nucleotide sequences of functional clones of the AIDS virus.

To examine the mechanism of lymphocytotoxicity induced by human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), an in vitro model has been developed. Introduction of an HTLV-III/LAV proviral clone, HXB2, into normal lymphocytes results in the production of virions and cell death. The complete nucleotide sequence of the proviral form of HXB2 has now been determined. Its structure is quite similar to that previously determined for HTLV-III/LAV clones whose biological capacities had not previously been demonstrated. The biological function of two additional clones of HTLV-III/LAV, BH10 and HXB3, are reported. Clone BH10 which lacks the 5' long terminal repeat sequences (LTR) and a portion of the 3' LTR is reconstituted by substituting the corresponding sequences of HXB2 and is shown to be capable of generating infectious cytopathic virions. Clone HXB3, which has been partially sequenced, is also found to be capable of producing lymphocytopathic virus. Clone HXB3 differs from HXB2 in its lack of a termination codon in 3' orf, demonstrating that 3' orf plays no major role in virus replication or cytopathic activity. These data provide the necessary background to allow the identification of viral determinants of replication, cytopathic activity, and antigenicity using these functional proviral clones.

Base Sequence↗
PubMed1990

Serial passage of infectious transcripts of a cucumber mosaic virus satellite RNA clone results in sequence heterogeneity.

Populations of the D-satellite RNA (D-sat) of cucumber mosaic virus (CMV) have a major site of heterogeneity at which a significant proportion of the molecules vary from each other. The generation of this heterogeneity was investigated by using infectious transcript RNA of a prototype D-sat clone as an inoculum of uniform sequence to initiate serial passages in five different host plant species. RNase protection assays indicated that heterogeneity at the same site seen in natural D-sat RNA populations developed within two to four passages in most of the hosts tested, and the proportion of the population exhibiting this heterogeneity accumulated to a much higher level in tobacco than in the other hosts. Characterization of the heterogeneity which developed in tobacco showed it to be qualitatively different from that in the original D-sat population. This analysis of a CMV satellite RNA is the first example in which infectious transcript RNA from a clone has been used to investigate the rapid generation and selection of heterogeneity in a genomic RNA population replicating in plants.

Base Sequence↗
PubMedDate not supplied

Sequencing of coronavirus IBV genomic RNA: a 195-base open reading frame encoded by mRNA B.

DNA sequencing of genomic cDNA clones of avian infectious bronchitis virus (IBV) has been carried out. 770 bases have been determined which include genomic sequences spanning the 5' termini of the two smallest mRNAs of the 3'-coterminal "nested" set: mRNA A and mRNA B. This region contains the complete coding sequences for mRNA B which are additional to those present in mRNA A. Two open reading frames are present, predicting proteins of Mrs 7500 and 9500.

Animals↗
PubMed1998

Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line.

Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13-->L). Both tat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13-->L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.

Amino Acid Sequence↗
PubMed1994

Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.

The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus.

The complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M(r) 92K and which is the putative RNA-dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in cell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3' coterminal subgenomic (sg) RNAs, the 5' termini of which have been mapped. The two sg RNAs are 2155 (sg1) and 934 (sg2) nt in length. ORF3 is expressed from sg1 RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sg1 RNA.

Amino Acid Sequence↗
PubMed2001

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6.

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Amino Acid Sequence↗
PubMed2000

Responses of cloned rainbow trout Oncorhynchus mykiss to an attenuated strain of infectious hematopoietic necrosis virus.

The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification.

Animals↗
PubMed2006

Recovery of infectious bluetongue virus from RNA.

Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.

Animals↗
PubMed2004

Human immunodeficiency virus type 1 infection of human brain-derived progenitor cells.

Although cells of monocytic lineage are the primary source of human immunodeficiency virus type 1 (HIV-1) in the brain, other cell types in the central nervous system, including astrocytes, can harbor a latent or persistent HIV-1 infection. In the present study, we examined whether immature, multipotential human brain-derived progenitor cells (nestin positive) are also permissive for infection. When exposed to IIIB and NL4-3 strains of HIV-1, progenitor cells and progenitor-derived astrocytes became infected, with peak p24 levels of 100 to 500 pg/ml at 3 to 6 days postinfection. After 10 days, virus production was undetectable but could be stimulated by the addition of tumor necrosis factor alpha (TNF-alpha). To bypass limitations to receptor entry, we compared the fate of infection in these cell populations by transfection with the infectious HIV-1 clone, pNL4-3. Again, transfected progenitors and astrocytes produced virus for 7 days but diminished to low levels beyond 8 days posttransfection. During the nonproductive phase, TNF-alpha stimulated virus production from progenitors as late as 5 weeks posttransfection. Astrocytes produced 5- to 20-fold more infectious virus (27 ng of p24/10(6) cells) than progenitors at the peak of 3 days posttransfection. Differentiation of infected progenitors toward an astrocyte phenotype increased virus production to levels consistent with infected astrocytes, suggesting a phenotypic difference in viral replication. Using this cell culture system of multipotential human brain-derived progenitor cells, we provide evidence that progenitor cells may be a reservoir for HIV-1 in the brains of AIDS patients.

Astrocytes↗
PubMed1991

An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells.

Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.

Base Sequence↗
PubMed2004

A convenient method for positive selection of retroviral producing cells generating vectors devoid of selectable markers.

Early retroviral vectors containing both a therapeutic gene and a dominant selectable marker gene, offered some distinct advantages with respect to gene therapy, in that they simplified the generation, isolation, and titration of retroviral producer cell clones, as well as the evaluation and selection of successfully targeted cells. However, a number of problems were engendered by this strategy: the promoter driving the selectable marker gene could interfere with transcription of the therapeutic gene, and immune responses could be induced to cells expressing foreign proteins of selection marker origin. Simplified retroviral vectors, which lack a selection marker gene, were constructed to address these problems, but the inability to use a selection marker has made identification and cloning of virus producing transfected cells a heavy burden. To maintain the benefits of simplified retroviral vectors, while providing a facile means to select packaging cells transfected with retroviral DNA, we cloned the bacterial selection marker gene encoding neomycin phosphotransferase (neo) into the plasmid backbone of the vector, but outside of the provirus, resulting in efficient selection of transfected packaging cells and generation of packaged virus which lacks the neo gene. This novel approach generates greater numbers of high infectious titer producing clones, after selection in G418 media, than does a co-transfection approach, due to integration of higher construct copy numbers per cell. No transmission of the selection marker gene to target cells was observed following retroviral transduction. Thus, our strategy eliminates the adverse consequences of a selection-based method, while diminishing the burden of identification of packaging cells transfected with vectors devoid of selectable markers.

DNA, Viral↗
PubMed2003

Cloning and nucleotide analysis of segment A gene of infectious bursal disease virus detected in Korea.

A strain of infectious bursal disease virus (IBDV) was detected from bursal tissues of chicks which suffered from infectious bursal disease (IBD) in Chinju, Korea and provisionally named as Chinju strain. A full-length cDNA clone for segment A gene of the virus was constructed, and complete nucleotide sequence of the gene including noncoding region was determined and analyzed by comparison with that of other IBDV strains. The segment A gene of Chinju strain consisted of 3,269 nucleotides including 862 adenine (26.4%), 917 cytosine (28.0%), 854 guanine (26.1%) and 636 thymine (19.5%). There were regions for two open reading frames (ORFs), ORF1 encoding the VP5 with ATG codon at nucleotides 98-100 and ORF2 encoding the polyprotein of VP2, VP4 and VP3 in the nucleotides 132-3,170. In deduced translation the ORF2 encoded 1,012 amino acids. The full nucleotide sequence of segment A gene and amino acid sequence of ORF2 of the Chinju strain showed 98-99% homology with those of the very virulent IBDVs (vvIBDVs) such as HK46, OKYM, UK661, UPM97/ 61, D6948 and BD3/99. In phylogenetic analysis of nucleotide and amino acid sequences, the Chinju strain was also related closely to the vvIBDVs. Hence, it was suggested that the Chinju strain is a vvIBDV. The nucleotide and amino acid sequences of the Chinju strain with pertinent information can be useful for the development of genetically engineered vaccines and diagnostic reagents against vvIBDV.

Amino Acid Sequence↗
PubMed1996

The 5' terminal sequence of alfalfa mosaic virus RNA 3 is dispensable for replication and contains a determinant for symptom formation.

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AIMV), can be infected with RNA 3 of the tripartitite AIMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter and nos terminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5' terminal 79 nucleotides (nt). When the sequence corresponding to the 5' terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5' terminal deletion of 79 nt was produced. The 5' truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.

Alfalfa mosaic virus↗
PubMed1983

Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.

Chromosome Deletion↗
PubMed1993

Structural and functional characterization of rev-like transcripts of equine infectious anemia virus.

Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.

Amino Acid Sequence↗
PubMed1986

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.

Animals↗