We have examined the parameters that determine the length and distribution of products synthesized processively by the human immunodeficiency virus reverse transcriptase (HIV-RT). On native or homopolymer templates, the overall length distribution of processively synthesized products is increased by increased temperature or deoxynucleoside triphosphate concentration, or decreased ionic strength. Specific terminations of processive synthesis on either native DNA or RNA templates occur most frequently at positions where the reverse transcriptase (RT) pauses during synthesis. These sites correlate with the template sequence 3'-(A/U)(A/U)(G/C)-5', particularly when this sequence is predicted to be base paired with another region of the template in a secondary structure. Many positions of termination are in similar positions on DNA or RNA templates. Notable exceptions are runs of A residues, which promote termination on DNA but not RNA templates. Termination intensities vary when different RTs are used demonstrating an influence of RT structure.
"In the case of set #2 (Figure 1C), premature terminations are apparent (with product lengths of mostly 47, 40 and 36-nt), whereas with set #1 these longer premature terminations are less evident (see also below, Figures 2A and 4A, regarding set #1). These results support the concept that the extent of RT-dependent processivity and, consequently pausing, can depend, for a given RT, on the sequences of the copied templates, for example, (12,13). "
[Show abstract][Hide abstract] ABSTRACT: We present evidence that the reverse transcriptase (RT) of human immunodeficiency virus type-1 stabilizes in vitro very short (2-nt) duplexes of 3'-overhangs of the primer strand that are annealed to complementary dinucleotides tails of DNA or RNA template strands, provided that these sequences contain at least one C or G. This RT-induced strand 'clamping' activity promotes RT-directed DNA synthesis. This function is achieved only when the functional template strand is adjacent to a second DNA or RNA segment, annealed upstream to most of the primer (without gaps). The combined clamp/polymerase activity is typical to RTs, as it was found in different RTs from diverse retroviral groups, whereas cellular DNA-polymerases (devoid of 3'→5' exonucleolytic activity) showed no clamp activity. The clamp-associated DNA-binding activity is markedly stabilized by dGTP, even when dGTP is not incorporated into the nascent DNA strand. The hereby-described function can help RTs in bridging over nicks in the copied RNA or DNA templates, encountered during reverse transcription. Moreover, the template-independent blunt-end synthesis of RTs can allow strand transfers onto compatible acceptor strands while synthesizing DNA. These RT properties can shed light on potentially-new roles of RTs in the reverse-transcription process and define new targets for anti-retroviral drugs.
Nucleic Acids Research 09/2010; 39(3):1042-53. DOI:10.1093/nar/gkq786 · 9.11 Impact Factor
"Expression of lentivirus accessory genes requires splicing or internal ribosome entry sites, both of which are defined by specific secondary structure in the RNA. These stable RNA structures may facilitate recombination by decreasing processivity of the viral reverse transcriptase (DeStefano et al., 1992). The outcome of most strand switches is not detected in natural infection because many recombinant progeny will be replication incompetent. "
[Show abstract][Hide abstract] ABSTRACT: Recombination contributes significantly to diversity within virus populations and ultimately to viral evolution. Here we use a recently developed statistical test to perform exploratory analysis of recombination in fourteen feline immunodeficiency virus (FIVpco) genomes derived from a wild population of cougars. We use both the global and local Phi statistical test as an overall guide to predict where recombination may have occurred. Further analyses, including similarity plots and phylogenetic incongruence tests, confirmed that three FIVpco lineages were derived from recombinant events. Interestingly, the regions of mosaic origin were clustered in the area encoding lentiviral accessory genes and largely spared the viral structural genes. Because some of the mosaic strains are currently geographically disparate, our data indicate that the dispersal of cougars infected with these strains was preceded by recombination events. These results suggest that recombination has played an important role in the evolution of FIVpco for this wild population of cougars.
[Show abstract][Hide abstract] ABSTRACT: Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration on the ejection of DNA from bacteriophage lambda. There is a concentration, N0, at which the minimum amount of DNA is ejected. At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior is due to the overcharging of DNA at high concentration of Mg⁺² counterions. As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive. N0 corresponds to the concentration at which DNA is neutral. Our theory fits experimental data well. The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide. Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory. They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions. Three problems in understanding the capsid assembly for a retrovirus are studied: First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied. We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane, which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well. Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied. The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity. For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.
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