Abstract
BackgroundIntersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.ResultsFollowing a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.ConclusionThese findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.
Highlights
Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic
This study examined the possible influence of small interfering RNA (siRNA) inhibition on HIV-1 replication in a dual infection and how siRNA may be used as a tool to enrich for HIV-1 recombination in specific regions of the HIV-1 genome
In summary, we have developed a method to rapidly enrich for HIV-1 recombinants by blocking each of two parental HIV-1 isolates in a dual infection with strainspecific siRNAs
Summary
Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. The consequences of intersubtype recombination within dual/superinfected individual can be profound and can lead to the immediate selection of unique recombinant forms (URFs) or subsequent transmission of stable circulating recombinant forms (CRFs) [9]. In East Africa, intersubtype A/D, A/C, and D/C recombinant forms are almost as common as the parental subtype A, C, and D [8]. These URFs and CRFs have the potential to foil vaccine strategies based on single subtypes and even lead to rapid drug resistance
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