Inter-strand homologous recombination of HIV-1 occurs mainly during minus strand proviral DNA synthesis. Recombination enhances viral diversity and facilitates HIV-1 escape from antiviral selective pressures including HAART 5; 9; 41
. The dipeptide fingers domain insertion are positioned at the end of the HIV-1 RT fingers domain and in the cleft where the primer-template hybrid binds, but far from the polymerase active site 27; 42
. Interestingly, although they are located outside of the polymerase active site, these mutations are associated with viral resistance to multiple NRTIs such as AZT, which bind to the polymerase active site 8; 17
HIV-1 RT interactions with the double stranded region of the template annealed to the primer were extensively studied 43
. However, RT interactions with the single stranded portion of the template are less well understood. Moreover, the structure of the tertiary complex of HIV-1 RT and a template with extended single stranded portion is not available. Positioning of the single stranded part of the template at the cleft near the tip of the fingers domain has been predicted but not proven.
Significantly, the fingers domain of HIV-1 RT undergoes a large conformational change during T/P binding (RT-T/P binary complex formation) 27
, and it is highly likely that the β3–β4 loop at the tip of the fingers domain is structurally dynamic during DNA synthesis. The β3–β4 loop is likely the section of the replicating RT molecule that first contacts RNA template secondary structures. 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.
The biochemical data in this report demonstrate that the SG insertion RT at the β3–β4 loop has altered RNA binding kinetics of HIV-1 RT, which also is likely to relate to the enhancement of the strand transfer activity. More importantly, the post-drug RT displayed a faster initial binding rate (kon
) than the pre-drug RT, leading to 7X time higher template binding affinity (KD
) than the pre-drug RT and 3X higher than the post-drug
SG RT. Thus, these biochemical data supports the idea that the SG fingers domain insertion elevates the transfer activity presumably by enhanced interaction to the RNA templates.
Enhanced binding of post-drug RT to the T/P could be also related to a mechanism previously proposed for AZT resistant mutations found at the connection domain of HIV-1 RT: the connection mutations stabilize the RT-template complex yielding more time for the bound RT molecule to remove the incorporated AZTMP at the 3’ end of the primer 45
. Similarly, the dipeptide insertion mutations may also confer AZT resistance by enhancing the binding affinity, which lengthens the time span for HIV-1 RT to remain bound to the 3’ end of the primer for pyrophosphorolysis of AZTMP. We also envision that the increased frequency of binding events of post-drug RTs to the T/P can increase the degradation capability of the RNA template by the RT polymerization-dependent and –independent RNase H activity, which in turn would enhance the strand transfer activity. Importantly, our data demonstrate the removal of the dipeptide insertion from the mutant concomitantly induces loss of both the AZTMP pyrophosphorolysis and elevated strand transfer. This supports the idea that AZT resistance and elevated strand transfer share a common mechanism, which is the enhanced interaction with the RNA template.
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 , the pre-drug RT showed a higher polymerization-dependent RNase H activity and a faster binding rate (kon
) to the template (), compared to pre-drug RT. These two biochemical alterations by the dipeptide insertion can mechanistically contribute to the enhanced strand transfer efficiency of the post-drug RT: the post-drug RT creates 1° cleavage of the donor template more efficiently, followed by faster 2° cleavage of the donor template, compared to the pre-drug RT. These elevated 1° and 2° cleavages during DNA synthesis will generate more gaps, which serve as evasion sites for the acceptor RNA template during the template switch, and ultimately increase the strand transfer activity. Importantly, the fast binding rate (kon
) of the post-drug RT can facilitate the 2° cleavage of the donor template, which likely requires the rebinding of RT after the 1° cleavage. Importantly, the overall delayed template degradation of the post-drug RT () is the result of both polymerization-dependent and independent RNase H activities. As shown in Supplementary figure 7
, the post-drug RT actually has reduced polymerization-independent RNase H activity, compared to the pre-drug RT. Thus, these two sets of data suggest that the polymerization-independent RNase H activity, which could be responsible for the delayed template degradation, is not mechanistically involved in the elevated strand transfer activity of the post-drug RT. Instead, the polymerization-dependent RNase H activity, which creates the invasion sites for the acceptor during DNA synthesis, is more likely the mechanistic reason for the elevated strand transfer activity of the post-drug RT.
In summary, the multiple drug resistant post-drug RT displayed altered biochemical behaviors including elevated strand transfer activity, increased polymerization-dependent RNase H activity and tighter binding affinity to the T/P substrate. We believe that with these simultaneous mechanistic changes made by the dipeptide insertion RT, a follow-up virological experiment to test the actual impact of the post-drug RT mutations on the recombination efficiency of the viruses should be conducted in the future.