The nature of the N-terminal amino acid residue of HIV-1 RNase H is critical for the stability of reverse transcriptase in viral particles

Abstract

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.

Importance: HIV-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.

FIG 1

The N-terminal residue of HIV-1 RNase H is highly conserved. The sequence of the protease cleavage site between RT (p51) and RNase H was analyzed for 1,850 isolates of HIV-1 and chimpanzee simian immunodeficiency virus present in the Los Alamos HIV sequence database (http://www.hiv.lanl.gov) using the web alignment tool. The amino acid that corresponds to the conserved sequence is shown at the bottom. Arrows indicate the protease cleavage site between RT and RNase H. The sequence logo at the top was generated using WebLogo (http://weblogo.berkeley.edu).

FIG 2

Changing the N-terminal residue of HIV-1 RNase H leads to the instability of RT and integrase in the virus particle. (A) Equivalent amounts of WT and RNase H N-terminal mutant virions (according to normalization of the amount of p24) were analyzed by Western blotting following production in 293T cells using helper plasmids and concentration by ultracentrifugation. Particles were probed with RT (top), integrase (middle), and p24 (bottom) antibodies. (B) Extended exposure of the immunoblots from panel A. (C) 293T cells transfected with helper plasmids producing WT or RNase H mutant virions were lysed 48 h after transfection, and the protein contents of equivalent amounts of cells were analyzed by immunoblotting with the indicated antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 3

Effect of N-terminal RNase H mutations on HIV-1 infectivity. Jurkat cells were infected with equivalent amounts (according to normalization of the amount of p24) of VSVg-pseudotyped WT or mutant RNase H mutant HIV-1 vectors. Infectivity was measured by flow cytometry at 3 days postinfection. The MOI for the WT was measured as indicated in the Materials and Methods section.

FIG 4

Intravirion processing of Gag and Gag-Pol polyproteins in HIV-1 RNase H N-terminal mutants. The WT and two representatives of RNase H mutant HIV-1 virion particles were produced in the presence of various concentrations of the HIV-1 protease inhibitor ritonavir, and the cleavage patterns of the Gag and GagPol polypeptides were analyzed by immunoblot analysis. The WT, as well as methionine (Met) and leucine (Leu) RNase H mutants, were probed with antibodies to RT (top) or p24 (bottom).

FIG 5

Constructs used for Vpr complementation and in vitro studies. The position and identity of RNase H N-terminal substitutions are indicated. The frameshift causing a mutation in pRK5-GagPol-FS is shown. PC, protease cleavage site between viral protease and RT; RH, RNase H.

FIG 6

Analysis of Vpr-RT-integrase-complemented HIV-1 virions. HIV-1 virions with GagPol containing WT (B) and RNase H N-terminal Leu (A) or Pro (C) mutants were produced in 293T cells with helper plasmids in the presence or absence of expression plasmids encoding Vpr-RT-integrase fusion proteins with WT or N-terminal RNase H mutant RT. Concentrated virions were normalized for their p24 contents by ELISA and analyzed by Western blotting using Vpr (top), RT (middle), and p24 (bottom) antibodies.

FIG 7

Infectivity of Vpr-RT-integrase-complemented WT or RNase H N-terminal mutant viruses. Jurkat cells were infected with equivalent amounts (according to normalization of the amount of p24) of WT (A) or N-terminal RNase H Leu (B), Met (C), Pro (D), or Trp (E) mutant VSVg-pseudotyped HIV-EGFP containing WT or RNase H mutant Vpr-RT-IN fusion proteins. Infectivity was determined by flow cytometry at 3 days postinfection. The MOI for the WT was measured as described in Materials and Methods.

FIG 8

In vitro analysis of the role of viral protease in the degradation of RNase H mutants. pRK5-GagPol-FS was used for in vitro transcription translation by use of an RRL system. Translation products were incubated at 30°C for 2 h with HIV-1 protease or phosphate buffer and analyzed via immunoblotting. Arrows, the identity of the fragments detected with RT and p24 antibodies. The antibodies used for each blot are indicated in bold with an α prefix.

FIG 9

The RNase H N-terminal residue is critical for RT subunit association. (A) 293T cells were cotransfected with Vpr-pro50-p51-IRES-p66 plasmids encoding WT or proline RNase H mutant p66 and ΔNRF D25A. Vpr-p51Δp66 was used as a control. Co-IP was performed on cell extracts using Vpr antibody and protein G beads. (B) 293T cells were cotransfected with Vpr-pro50-p51-IRES-p66 plasmids encoding WT or different RNase H mutant p66 proteins and ΔNRF D25A. Vpr-p51Δp66 was used as a control. Co-IP was performed on cell extracts using Vpr antibody and protein G beads. The antibodies used for each blot are indicated in bold with an α prefix. Lanes IP, immunoprecipitated proteins; lanes Inp, input proteins.

FIG 10

Intravirion analysis of the role of viral protease in the RNase H degradation mutants. HIV-1 virions with either protease-inactivating D25A mutant GagPol or RNase H N-terminal proline mutant GagPol (labeled GagPol Pro) were produced in 293T cells in the presence or absence of Vpr-pro50-p51-IRES-p66 plasmids carrying WT or RNase H N-terminal mutant p66. Concentrated virions were analyzed by Western blotting using the indicated antibodies following normalization of their amounts via p24 ELISA. The antibodies used for each blot are indicated in bold with an α prefix. Numbers on the left are molecular masses (in kDa).