Exogenous Reverse Transcriptase Research Articles

Novel Functions of Prototype Foamy Virus Gag Glycine- Arginine-Rich Boxes in Reverse Transcription and Particle Morphogenesis

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

BackgroundINI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication.ResultsWe found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate.ConclusionOur results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication.

Mutations of the RNase H C helix of the Moloney murine leukemia virus reverse transcriptase reveal defects in polypurine tract recognition.

Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged alpha-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (DeltaC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5' and 3' viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5' end.

The protease and reverse transcriptase of the tobacco LTR retrotransposon Tnt1 are enzymatically active when expressed in Escherichia coli.

The open reading frame (ORF) of the tobacco retrotransposon Tnt1-94 was over-expressed in Escherichia coli to assay its protease and reverse transcriptase (RT) enzymatic activities. In E. coli, Tnt1-94 polyprotein is cleaved off by the element-encoded protease to release a Gag protein with an apparent molecular mass of 37 kDa that forms high-density aggregates. The catalytic site of Tnt1-94 protease (D-T-A) as determined by deletion analysis differs from that of retroviruses and of well-characterized retrotransposons (D-T/S-G). The cleaved or uncleaved ORF of Tnt1-94 displays an exogenous RT activity. Over-expression of plant retrotransposons ORFs in E. coli provides a very useful strategy to assay the enzymatic activities of their proteins and to determine their catalytic sites.

Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle.

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

Role of Vif in human immunodeficiency virus type 1 reverse transcription.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif mutant HIV-1 virions are defective in their ability to synthesize proviral DNA in vivo. Here, we examine the role of Vif in viral DNA synthesis in the endogenous reverse transcriptase (RT) reaction, an in vitro assay in which virions synthesize viral DNA by using endogenous viral RNA as a template. vif mutant virions showed a significant reduction in endogenous RT activity despite similar levels of exogenous RT activity. Analysis of the viral DNA products on agarose gels demonstrated that this reflects reduced synthesis of short minus- and plus-strand DNA products in addition to those of full genomic length. Quantitative PCR analysis of endogenous reverse transcription provided further evidence for reduced formation of both initial and completed reverse transcripts. Vif had no effect on genomic RNA dimerization or the stability of the RNA dimer linkage. These results suggest that Vif is important for an early event after virus entry but preceding or during the early stages of viral DNA synthesis. This may be due to an intrinsic effect on reverse transcription or a preceding postentry event(s), such as virion uncoating or disassembly of the virion core. Drugs targeted to Vif function may provide a new therapeutic approach to inhibiting HIV-1 reverse transcription.

Kinetics of inhibition of endogenous human immunodeficiency virus type 1 reverse transcription by 2',3'-dideoxynucleoside 5'-triphosphate, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thion e, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives.

Recently, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. They interact with the reverse transcriptase of HIV-1 in a way different from that of previously studied reverse transcriptase (RT) inhibitors. We established an endogenous RT assay, starting from intact HIV-1 virions. This assay mimics the reverse transcription process in the HIV-infected cell more closely than RT assays with artificial templates. We investigated the inhibition of endogenous HIV-1 reverse transcription by the TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (R-82150) in comparison with the HEPT derivative 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and 2',3'-dideoxyguanosine 5'-triphosphate. The kinetics and characteristics of RT inhibition by TIBO in the endogenous RT assay were similar to those found previously for the exogenous RT assay (following addition of exogenous template/primer); thus, RT inhibition by TIBO was specific for HIV-1 and the extent of RT inhibition was dependent on which of the four substrates (dATP, dTTP, dGTP, and dCTP) was present in limited concentrations. Of the three enzymatic activities, RNA-dependent DNA polymerization was preferentially inhibited, and inhibition was not competitive with respect to the natural substrates. HIV-1 RT behaved as an allosteric enzyme, which means that positive cooperativity for binding of the substrate was observed. TIBO behaved as an allosteric inhibitor by causing a concentration-dependent decrease in this cooperativity.

Inhibition of Human Immunodeficiency Virus Type I Reverse Transcriptase by Suramin-related Compounds

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

Accumulation and breakdown of RNA-deficient intracellular virus particles in interferon-treated NIH 3T3 cells chronically producing Moloney murine leukemia virus.

Interferon treatment of NIH 3T3 cells chronically infected with Moloney murine leukemia virus inhibited about 95% of virus release. This inhibition was accompanied by a three- to twofold accumulation of intracellular virions. However, this accumulation could be demonstrated only be exogenous reverse transcriptase reaction assay or radioactive labeling of the assembled viral proteins. It could not be shown by the endogenous reverse transcriptase reaction assay, which depended on endogenous viral RNA, or by labeling the encapsidated viral RNA. It was therefore evident that most of the intracellular virions accumulated in interferon-treated cells were RNA deficient. Hybridization analysis revealed that these virions were deficient of genomic viral RNA, whereas size analysis by gel electrophoresis suggested that the deficiency of 4S RNA normally packaged in Moloney murine leukemia virus was even stronger. Our data also suggested that this RNA deficiency was not due to degradation of the encapsidated RNA, but more likely to a defect in virus assembly. RNA-lacking intracellular virions were unstable; they were found to collapse before being released.

Native ribonucleoprotein is an efficient transcriptional complex of avian myeloblastosis virus.

A native ribonucleoprotein (RNP) complex of avian myeloblastosis virus was prepared under conditions that gave optimal cDNA synthesis. The complex was an autonomous transcriptional unit capable of synthesizing DNA complementary to the RNA virus genome in the absence of exogenous reverse transcriptase (RNA-dependent DNA nucleotidyltransferase), genomic RNA, and primer. The RNA of the RNP complex cannot be translated in an in vitro cell-free translational system. The RNP contains intact viral RNA, the two subunits of the reverse transcriptase (beta and alpha), the p32 polypeptide resulting from the cleavage of the beta subunit into the alpha subunit, and p12. The principal polypeptide constituent of the RNP complex is the highly basic protein p12, which occurs at a molar ratio of 40:1 in relation to the beta subunit of the polymerase. When examined by the electron microscope, the RNP complex appears similar to the beaded structure of chromatin fiber. A significant portion of these molecules are circular, with headlike structures attached. The circular nature of the proviral DNA and the ability of the RNP complex to generate large intact cDNA copies from the natural primer end suggest that the 5' and 3' ends of the viral RNA are in proximity when in the RNP complex.

RNA viraux des cellules infectées par un retrovirus murin : (II) influence de la toyocamycine sur le métabolisme des RNA viraux

Nous avons précédemment montré que la Toyocamycine (analogue de l'adénosine), ajotée à des cultures de cellules produisant le virus de Friend provoque une diminution de la production virale et une perte de l'infectiosité. On observe également que le RNA 60–70 S des virions incorpore de la Toyocamycine. Dans ce travail, nous avons examiné l'état des RNA viraux intracellulaires. 16 heures après le début d'un traitement par une dose cytostatique de Toyocamycine (0.2 g/ml), on trouve par hybridation moléculaire que les cellules contiennent les 3/4 des RNA viraux présents dans les cellules non traitées. Ces RNA se répartissent en trois espèces majeures 70S, 35S et 20–24S. Lorsque les hybrides cDNA-RNA sont analysés sur une colonne d'hydroxyapatite, le cDNA viral est hybridé à 100 p. cent avec les RNA contenant la Toyocamycine. Par contre, l'analyse des mêmes hybrides par la nucléase S 1 montre que 90 p. cent du même cDNA est protégé par des concentrations équivalentes de RNA. Cette différence entre les deux méthodes d'hybridation suggère que la Toyocamycine déstabilise localement les régions où elle a été incorporée et les rend sensibles à la nucléase S 1 . Enfin, la teneur en Poly (A) des RNA viraux n'est pas significativement modifiée. Tous ces faits indiquent que l'inactivation des virus résults de l'incorporation de la Toyocamycine dans de RNA viral. The adenosine analogue Toyocamycin (4 amino-5-cyano 7 B-D ribofuranosyl-pyrrolo 2-3-d pyrimidine) is known to incorporate into the RNA species of mammalian cells. However, structural changes owing to the incorporation of the drug prevent the maturation of preribosomal 45S RNA precursor into 28 and 18S ribosomal RNA. The drug does not impede the transcription of other cellular RNA species such as transfer RNA, 5S ribosomal RNA, other small RNA species and does not prevent occurence of rapidly labeled RNA in cytoplasmic polysomes. We have previously shown that Toyocamycin depresses the production of a murine retrovirus by a chronically infected cell line (Eveline cells producing the Friend virus complex). Although possessing a 70S RNA and an exogenous reverse transcriptase activity the viruses released under Toyocamycin treatment are not infectious and lack endogenous reverse transcriptase activity. This can be explained by the incorporation of Toyocamycin in the viral RNA, resulting in alteration of transcriptional and translational capacities. In this work we examined the effect of Toyocamycin on intracellular viral RNA synthesis. Molecular hybridization techniques using a cDNA probe prepared with purified murine virus revealed that after treatment for 16 hours with a cytostatic concentration of Toyocamycin (0,2 g/ml), cells were still containing 75 per cent of the viral RNA present in untreated cells. Hybrids between the cellular RNA species and viral cDNA were analysed by chromatography on hydroxyapatite or by S 1 nuclease treatment. It was observed that the cDNA was totally hybridized when the first method was used, whereas 90 per cent only were protected against digestion by S 1 nuclease. The difference observed between the two methods suggests that the hybrid regions containing the analogue are somewhat destabilized and become more sensitive to the S 1 nuclease action. The intracellular viral RNA species are distributed into three main classes sedimenting at 70S, 35S and 20-24S. This profile does not show great differences with that of untreated infected cells. It is assumed that the 70S RNA comes from viruses budding at the plasmic membrane and from intracellular vacuolar particles. When the Toyocamycin-containing RNA is heated at 65°C before loading onto the sucrose gradient, most of the hybrids formed with viral cDNA are located in a peak sedimenting at 20S–24S. This result can be compared to the one observed with the 70S RNA isolated from extracellular virions synthezised in the presence of Toyocamycin, as it is dissociated after heating into 35S and 20–24 S species. It is not yet clearly established whether the 20–24S RNA detected by hybridization with the viral cDNA is a subgenomic fraction of viral RNA rather than a degradation product of 35S due to the incorporation of toyocamycin. Further experiments are currently performed to test the translational capacities of these Toyocamycin-containing viral RNA.

Preparations enriched for “immature” murine leukemia virus particles that remain in tissue culture fluids are deficient in Pr65 gag proteolytic activity

Partially purified Moloney or Rauscher murine leukemia virus preparations obtained from 24-hr harvests of tissue culture fluids were separated into Pr65 gag enriched and Pr65 gag deficient fractions by centrifugation on shallow sucrose or sucrose-D 2O density gradients, e.g., 30 to 50% sucrose (w/v). The Pr65 gag enriched fraction had a slightly higher relative density (ϱ = 1.18–1.20 g/cc) and a greatly increased number of “immature” virions (30%), than the Pr65 gag deficient fraction (ϱ = 1.16–1.18 g/cc; 2% immature virions). These two morphologically distinct virus populations were then further characterized as to the state of the RNA, the relative amount of exogenous reverse transcriptase activity, and the presence or absence of Pr65 gag proteolytic activity. Both types of particles possessed 70 S RNA and similar amounts of exogenous reverse transcriptase activity. However, only the Pr65 gag deficient particles exhibited any Pr65 gag proteolytic activity when incubated with Nonidet P-40 (2%, w/v), 10 m M DTT, and a Pr65 gag substrate, i.e., immature virions. Thus it appears that the immature Rauscher and Moloney leukemia virions that remain in tissue culture fluids of chronically infected cells possess a cleavable Pr65 gag, but are deficient in Pr65 gag proteolytic activity.