Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits.
IMPORTANCEHIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process.
Like all retroviruses, human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, synthesizes and packages its main structural and enzymatic proteins as precursor polyproteins. For HIV-1, these polyproteins are p55 (Gag) and p160 (GagPol). Gag is the most abundant polyprotein and is translated from a genome-length mRNA that contains the Gag and GagPol open reading frames. The synthesis of GagPol requires a ribosomal frameshift leading to a Gag/GagPol ratio of about 20:1 in the virus particle (1). Individual mature viral proteins are generated following viral assembly as a result of a series of proteolytic cleavage events at specific positions catalyzed by the viral protease, which is synthesized as a part of GagPol (2).One protein that is released as a result of proteolytic processing of GagPol is reverse transcriptase (RT). RT catalyzes the reaction for the conversion of viral RNA to...