No abstract

Template switching during reverse transcription promotes recombination in retroviruses. Efficient switches have been measured in vitro on hairpin-containing RNA templates by a two-step mechanism. Pausing of the reverse transcriptase (RT) at the hairpin base allowed enhanced cleavage of the initial donor RNA template, exposing regions of the cDNA and allowing the acceptor to base pair with the cDNA. This defines the first or docking step. The primer continued synthesis on the donor, transferring or locking in a second step. Here we determine the enzyme-dependent factors that influence template switching by comparing the RTs from human immunodeficiency virus, type 1 (HIV-1), and equine infectious anemia virus (EIAV). HIV-1 RT promoted transfers with higher efficiency than EIAV RT. We found that both RTs paused strongly at the base of the hairpin. While stalled, HIV-1 RT made closely spaced cuts, whereas EIAV RT made only a single cut. Docking occurred efficiently at the multiply cut but not at the singly cut site. HIV-1 nucleocapsid (NC) protein stimulated strand transfers. It improved RNase H activity of both RTs. It allowed the EIAV RT to make a distribution of cuts, greatly stimulating docking at the base of the hairpin. Most likely, it also promoted strand exchange, allowing transfers to be initiated from sites throughout the hairpin. Minor pause sites beyond the base of the hairpin correlated with the locking sites. The strand exchange properties of NC likely promote this step. We present a model that explains the roles of RNase H specificity, template structure, and properties of NC in the two-step transfer reaction.

No abstract

The 2,3-dideoxy-3-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primertemplate, results in the lowered processivity. The mutant RTs exhibit normal DNA 3-end-directed and RNA 5-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations. HIV-13 reverse transcriptase (RT) is a heterodimer consisting of a 66-kDa subunit (p66) and a 51-kDa subunit (p51). The p51 and p66 subunits share a common N terminus; the p51 subunit lacks 120 amino acids from the C terminus of the p66 subunit. Active sites for polymerase and ribonuclease H (RNase H) reside in p66. The highly conserved motif YXDD in the polymerase active site of HIV-1 and other retroviral RTs is essential for polymerase activity (1). In HIV-1 RT, Met-184 is the second amino acid in this conserved YXDD motif (2). Two mutations, M184I and M184V, arise in HIV-1 RT after treatment with the RT inhibitor 2Ј,3Ј-dideoxy-3Ј-thiacytidine, or 3TC (3-5), and confer high level resistance to 3TC. 3TC is a nucleoside analog that lacks the 3-OH of the ribose ring; incorporation of 3TC blocks viral DNA synthesis. It is the L-pseudo sugar version of 3TC that is used to treat patients. The M184I/V mutations cause steric hindrance between the oxathiolane ring of 3TCTP and the ␤-branched side chain of valine or isoleucine, which hinders incorporation of 3TCTP (6 -8). Wild-type MLV RT has valine in the second position of the YXDD motif (Val-223), and wild-type MLV is resistant to 3TC (9). Replacing the valine at position X in the YXDD motif of the WT MLV RT with methionine (V223M) makes the virus more susceptible to 3TC, and the MLV RT more sensitive to 3TCTP (10). Both HIV-1 and MLV RTs with an alanine substitution at the X position are moderately resistant to 3TC in vitro, even though alanine is not a ␤-branched amino acid (10, 11). This suggests that other factors, such as altered positioning of the template prim...

Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digested in vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysomeassociated endonuclease.