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PubMed1995

Both virus and host components are important for the manifestation of a Nef- phenotype in HIV-1 and HIV-2.

While it has been demonstrated that the Nef protein of simian immunodeficiency virus is obligatory for the establishment of high viral loads and the development of simian AIDS in rhesus macaques, demonstrating a critical role for the human immunodeficiency virus (HIV) Nef protein in tissue culture has been elusive. Data have been contradictory as to whether Nef has a negative or positive influence on in vitro virus replication. In an attempt to define a role for Nef during virus propagation in tissue culture and to obtain virus-host systems that could distinguish between the Nef mutant and wild-type viruses, we have introduced mutations into the nef genes of infectious molecular clones of three HIV-1 strains and two isolates of the HIV-2ROD strain and have investigated the capacity of viruses derived from them to infect a number of CD4-positive T-cell lines and peripheral blood mononuclear cells (PBMC). Mutating the nef gene of all viruses had a modest negative effect on virus production in activated PBMC. In some T-cell lines with some viruses, the effects were severe, and little or no Nef mutant virus could be detected. In other cell lines, the result of mutating the nef gene either had no effect or had a slight negative effect on the replication kinetics. Therefore, whether the consequences of loss of Nef activity can be demonstrated in vitro depends on both the particular virus and the host cell used, suggesting that Nef is exerting its activity on some cellular pathway. In addition, we describe the construction and properties of hitherto unreported infectious molecular clones of the ROD strain of HIV-2.

Amino Acid Sequence↗
PubMed2004

Construction of an infectious HIV type 1 molecular clone from an African patient with a subtype D/C Recombinant Virus.

The majority of HIV-1 infections worldwide occur in Africa, where subtype B viruses are rare and intersubtype recombinants are common. Pathogenesis and vaccine studies need to focus on viruses derived from African patients, and infectious HIV-1 molecular clones can be useful tools. To clone non-B subtypes and recombinant viruses from patients, we cultivated HIV-1 from the plasma of a Kenyan long-term survivor. Viral DNA was cloned into a plasmid, which was transfected into COS cells; progeny virus was propagated in PBMCs. Sequence analyses revealed that both the patient's plasma HIV-1 RNA and the cloned DNA genomes were recombinants between subtypes D and C; subtype C sequences comprised the nef and LTR regions. The cloned virus used the CCR5 coreceptor and did not form syncytia in vitro. This infectious HIV-1 subtype D/C recombinant molecular clone obtained from a Kenyan long-term survivor promises to be useful to study pathogenesis and vaccine design.

Animals↗
PubMed1990

Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin.

An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for attenuated HIV, which could be used as a vaccine. We have reconstructed the mutation found in the E variants within the infectious HIV clone HXB-2 and demonstrated that the resulting virus retains its noninfectious phenotype.

Base Sequence↗
PubMed2004

Construction of a full-length human T cell leukemia virus type I genome from MT-2 cells containing multiple defective proviruses using overlapping polymerase chain reaction.

Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.

Cell Line, Transformed↗
PubMed2002

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.

Amino Acid Sequence↗
PubMed1989

Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs.

Full-length cDNA of the 6.3 kb turnip yellow mosaic virus (TYMV) genome was placed between a T7 promoter and a unique Hind III site. In vitro transcription of Hind III-linearized DNA of clone pTYMC yielded full-length RNA transcripts. In inoculations of Chinese cabbage protoplasts and plants, capped transcripts and virion RNA had similar specific infectivities and produced similar systemic symptoms. We have used the pTYMC clone in studies of the expression of two overlapping open reading frames (1.9 kb and 5.5 kb ORFs) by making mutants with alterations in the initiation codons. Evidence is presented from in vitro translations of mutant and wild type RNAs that both ORFs are expressed from TYMV RNA. A mutant in the initiation codon of the 5.5 kb ORF did not replicate in protoplasts, while mutants in the initiation codon of the 1.9 kb ORF replicated at low levels. The two groups of mutants were not able to complement each other.

Brassica↗
PubMed2004

Long-term persistence of infection in chimpanzees inoculated with an infectious hepatitis C virus clone is associated with a decrease in the viral amino acid substitution rate and low levels of heterogeneity.

Two chimpanzees, 1535 and 1536, became persistently infected following inoculation with RNA transcripts from cDNA clones of hepatitis C virus (HCV). Analysis of the HCV genomes from both animals showed an accumulation of amino acid substitutions over time. The appearance of substitutions in the envelope genes was associated with increased antienvelope antibody titers. However, extensive mutations were not incorporated into hypervariable region 1 (HVR1). A comparison of the nonsynonymous substitution rate/synonymous substitution rate was made at various time points to analyze selective pressure. The highest level of selective pressure occurred during the acute phase and decreased as the infection continued. The nonsynonymous substitution rate was initially higher than the synonymous substitution rate but decreased over time from 3.3 x 10(-3) (chimpanzee 1535) and 3.2 x 10(-3) (chimpanzee 1536) substitutions/site/year at week 26 to 1.4 x 10(-3) (chimpanzee 1535) and 1.7 x 10(-3) (chimpanzee 1536) at week 216, while the synonymous substitution rate remained steady at approximately 1 x 10(-3) substitutions/site/year. Analysis of PCR products using single-stranded conformational polymorphism indicated a low level of heterogeneity in the viral genome. The results of these studies confirm that the persistence of infection is not solely due to changes in HVR1 or heterogeneity and that the majority of variants observed in natural infections could not arise simply through mutation during the time period most humans and chimpanzees are observed. These data also indicate that immune pressure and selection continue throughout the chronic phase.

Amino Acid Substitution↗
PubMed2005

Construction and analysis of genomic, full-length infectious foamy virus DNA clones.

The molecular engineering of recombinant plasmid DNA clones containing the full-length and replication-competent feline foamy (retro)virus (FFV) proviral genome is described. The methods used to combine subgenomic FFV DNA fragments can be applied to other retrovirus genomes, resulting in full-length, bacterially cloned retroviruses. In addition, techniques used to determine the replication competence of the cloned viral genomes are described. Alternative technologies (not described here) are also discussed, together with the application of cloned genomes in modern molecular virology; for example, in functional genetic studies or as starting material for retrovirus vector development.

Animals↗
PubMed2000

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype.

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Amino Acid Sequence↗
PubMed1991

Phenotypic heterogeneity in a panel of infectious molecular human immunodeficiency virus type 1 clones derived from a single individual.

Previously, we and others have demonstrated a relation between the clinical course of human immunodeficiency virus type 1 (HIV-1) infection and biological properties of HIV-1 variants such as replication rate, syncytium-inducing (SI) capacity, and cytotropism. For the molecular analysis of the biological variability in these properties, we generated a panel of phenotypically distinct yet genetically highly homologous infectious molecular clones. These clones were derived from HIV-1 isolates, mostly recovered by direct clonal isolation, from a single individual in whom a transition from non-SI to SI isolates had been identified over time. Of 17 molecular clones tested, 8 were infectious. The clones exhibited differences in SI capacity and T-cell line tropism. Their phenotypes corresponded to those of their parental isolates, formally demonstrating that biological variability of HIV-1 isolates can be attributed to single molecular clones. With these clones we demonstrated that SI capacity and tropism for the H9 T-cell line, almost invariably coupled in primary HIV-1 isolates, are discernible properties. Also different requirements appeared to exist for H9 and Sup T1 cell line tropism. We obtained evidence that T-cell line tropism is not caused by differences in level of HIV-1 expression but most probably is restricted at the level of virus entry. Restriction mapping of four clones with divergent phenotypes revealed a high degree of nucleotide sequence homology (over 96.3%), indicating the usefulness of these clones for the tracking of genetic variability critical for differences in biological phenotype.

Acquired Immunodeficiency Syndrome↗
PubMed1997

A recombinant retroviral system for rapid in vivo analysis of human immunodeficiency virus type 1 susceptibility to reverse transcriptase inhibitors.

We have developed a new recombinant retroviral system in which a library of infectious molecular clones of human immunodeficiency virus type 1 (HIV-1) is constructed with reverse transcriptase (RT) genes derived from viral RNA sequences in plasma. HIV-1 RT is amplified from plasma HIV-1 RNA by nested RT-PCR and cloned into a RT-defective HIV-1 proviral vector (xxLAI-np), generating 10(3) to 10(4) recombinant proviral clones from each reaction. The bulk cloning products or individual molecular clones are transfected into MT-2 cells to generate infectious virus. The resultant viruses are assayed for drug susceptibility in CD4+ cell lines to determine either the dominant phenotype of the recombinant virus mixture or the phenotypes of the individual viral clones. DNA sequencing of the cloned RT genes can identify mutations associated with phenotypic resistance of clonal mixtures or individual clones. This method can be used to rapidly detect the in vivo emergence of HIV-1 quasispecies resistant to RT inhibitors.

Anti-HIV Agents↗
PubMed1997

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase-PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6-28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Animals↗
PubMed1992

Infectious defective interfering particles of VSV from transcripts of a cDNA clone.

The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported. Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones. The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions. No helper VSV was required. Replication occurred at high levels and was assayed by direct biochemical means. An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.

Base Sequence↗
PubMed1987

Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters.

The interspecies transmission of scrapie is frequently associated with exceptionally long incubation periods at first passage in the new host compared to later passages (the species barrier effect). The basis of this was investigated using the 139A strain of scrapie which had been cloned by three serial passages in mice at limiting infectious doses. Cloned scrapie was passaged through hamsters (twice) or rats (thrice) and then reisolated in mice. Large species barrier effects were encountered on mouse-to-hamster and hamster-to-mouse passage resulting in the isolation of a mutant strain, 139-H/M, with properties very different from 139A. In contrast, the strain reisolated from rats was indistinguishable from 139A. However, a large species barrier was encountered at the mouse-to-rat passage but not at the rat-to-mouse passage. It is suggested that the transmission of scrapie between species may be associated with no change in properties or a permanent change in the scrapie genome due to the selection of mutants. A third possibility, the donor species effect, is a temporary change occurring only at first passage in the new host species which is largely or entirely caused by the introduction of material from the previous host. We speculate that the donor species effect could be explained if some host protein forms a functional part of the infectious agent.

Animals↗
PubMed1993

Graffi murine leukemia virus: molecular cloning and characterization of the myeloid leukemia-inducing agent.

The Graffi murine leukemia virus (MuLV) is a retroviral mixture that induces predominantly myeloid leukemia in several inbred strains of mice. To analyze the viral component responsible for the myeloid leukemogenesis, we cloned several proviruses from a Graffi MuLV-infected cell line. Several infectious molecular clones were obtained that could be classified into two distinct groups of infectious MuLV. Both types of MuLV were nondefective, ecotropic, and NB tropic and induced granulocytic leukemia in BALB/c and NFS mice. Restriction enzyme analysis and molecular hybridization with several MuLV probes on one molecular clone from each group revealed that both groups are closely related to each other but are clearly distinct from all known retroviruses. One component of MuLV, however, induced leukemia with a shorter latency period and harbored a lengthier long terminal repeat. The long terminal repeat of the more leukemogenic component of MuLV had acquired a 60-bp perfect duplication in the U3 region. Analysis of the tumor DNAs with probes for the mouse T-cell receptor and immunoglobulin heavy chain genes revealed frequent rearrangements with one or both probes. This concomitant expression by leukemic cells of markers of different lineages, observed in human leukemias, has been termed "lineage infidelity" and confirms that the latter rearrangements are not restricted to hematopoietic precursors committed to lymphoid differentiation.

Animals↗