Factors That Determine the Efficiency of HIV-1 Strand Transfer Initiated at a Specific Site

Abstract

Summary HIV-1 employs strand transfer for recombination between the two viral genomes. We previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism, in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long distance propagation of the hybrid. The evaluation was performed on a unique substrate with a short gap serving as a pre-created invasion site (PCIS). In our current work, this substrate provided the opportunity to test what factors influence a specific invasion site to support transfer, and distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or -independent mode. Polymerization-dependent and -independent RNase H were found to be important to create efficiently-used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a PCIS in the presence of NC, were stimulated by polymerization-dependent but not -independent RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer, even if synthetic progress of the RT was intentionally slowed, which is consistent with strand exchange by random walk in which rate declines precipitously with distance.

Host SAMHD1 Protein Promotes HIV-1 Recombination in Macrophages

“…Upon pausing of RT during the DNA synthesis, the RNase H activity degrades the RNA templates. This creates ssDNA regions that can initiate the strand transfer and homologous recombination by the primer annealing to the homologous region of the other RNA genome harbored in the same viral particle (7,8). In this "strand invasion" mechanism model, factors that cause RT pausing directly affect recombination and strand transfer frequency.…”

Altered Strand Transfer Activity of a Multiple-Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant with a Dipeptide Fingers Domain Insertion

“…In addition, the stable interaction of HIV-1 RT with template, which is essential for efficient RNA template cleavage by the RNase H activity of RT, plays a key role in the template switching (recombination) activity of HIV-1 RT between two homologous RNA strands 4; 29; 30; 31 . Thus, we hypothesized that the SG dipeptide insertion found in the fingers domain affects the strand transfer activity of HIV-1 RT.…”

“…Figure 2 is a diagram depicting the strand transfer system used in our previous study 29; 30 . In this system, a 5’ 32 P-labeled 20 nt DNA primer is annealed to an 80 nt “donor” non-viral RNA sequence template 26 .…”

“…It has been shown that stable local structures of the RNA template mechanistically induce RT pausing 44 . Pausing during synthesis in turn facilitates the RNA template degradation by RNase H activity of RT, which is an essential step for strand transfer 29; 30 . Thus we hypothesized that the dipeptide insertion in HIV-1 RT, which lies at the tip of the fingers domain, may alter the RT-RNA template interaction and thus strand transfer efficiency of HIV-1 RT.…”

“…It is well established that the efficient RNA template degradation by RNase H increases the strand transfer of RT 29; 30; 36; 37; 42; 46 . As shown in Fig.…”