Abstract
Each of the HIV-1 pol-encoded enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN) are released during virion maturation and are active only as dimers. Of the three, only RT comprises subunits of different mass. RT in mature infectious virions is a heterodimer of 66 kDa and 51 kDa subunits, even though its gene encodes a 66 kDa protein. The RT p51 subunit is formed by HIV-1 PR-catalyzed cleavage of RT p66, resulting in the removal of a ribonuclease H (RNH) domain. Given the existence of completely active recombinant p66/66 RT homodimers and alternative RT oligomers in other retroviruses, the apparent need for p66/51 RT heterodimers in the HIV-1 virion is unclear. To determine why the generation of active viral RT requires three processing events, we introduced mutations in the p51-RNH and RT-IN protease cleavage sites of an infectious HIV-1 molecular clone. Mutation of the RT-IN cleavage site had no effect on the activity or proteolytic stability of the p98/51 RT product, although infectivity was severely attenuated. This result was similar to findings previously reported for the PR-RT cleavage site. Surprisingly, mutation of the internal p51-RNH cleavage site did not increase RT p66 content, but resulted in attenuated virus containing greatly decreased levels of RT that was primarily RT p51. We further identified a compensatory second-site mutation T477A, found to restore RT activity and processing to p66/51 RT when introduced in the background of p51-RNH cleavage site mutations. These studies demonstrate that cleavage of the internal p51-RNH junction, not the flanking N-terminal or C-terminal junctions is essential for proteolytic stability of functional RT during virion maturation. These findings further emphasize the importance of the RNH domain in regulating proteolytic generation of p66/51 RT. The overall need for the RT heterodimer is attributable to the generation of its subunits. Formation of the 51 kDa subunit or cleavage of the p51-RNH junction is essential for RT stability in the virion, whereas formation of the 66 kDa subunit is important for efficient viral replication.
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