The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site

Michael E Abram,Michael A Parniak,Stefan G Sarafianos

doi:10.1186/1742-4690-7-6

Abstract

BackgroundThe p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51↓RNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness was restored after repeated passage of mutant viruses, due to reversion of the mutated sequences to wild-type. However, in one case, the recovered virus retained the mutated p51↓RNH cleavage site but also developed an additional mutation, T477A, distal to the cleavage site. In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT.ResultsWhile the T477A mutation arose during serial passage only with the F440V mutant background, introduction of this substitution into a variety of RT p51↓RNH cleavage site lethal mutant backgrounds was able to restore substantial infectivity and normal RT processing to these mutants. T477A had no phenotypic effect on wild-type HIV-1. We also evaluated the impact of T477A on the kinetics of intravirion Gag-Pol polyprotein processing of p51↓RNH cleavage site mutants using the protease inhibitor ritonavir. Early processing intermediates accumulated in p51↓RNH cleavage site mutant viruses, whereas introduction of T477A promoted the completion of processing and formation of the fully processed RT p66/p51 heterodimer.ConclusionsThis work highlights the extraordinary plasticity of HIV-1 in adapting to seemingly lethal mutations that prevent RT heterodimer formation during virion polyprotein maturation. The ability of T477A to restore RT heterodimer formation and thus intravirion stability of the enzyme may arise from increased conformation flexibility in the RT p51↓RNH cleavage site region, due to loss of a hydrogen bond associated with the normal threonine residue, thereby enabling proteolytic cleavage near the normal RT p51↓RNH cleavage site.

Highlights

The p51 subunit of the Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation
Since HIV-1 virions contain essentially equivalent amounts of p66 and p51 RT subunits [17,18], proteolytic cleavage of the p51↓RNH junction may possibly be an important factor in the production of replication-competent virions. Both recombinant RT p66/ p66 homodimers and RT p66/p51 heterodimers show similar catalytic properties (DNA polymerase and RNH activities) in vitro [19,20,21], which begs the question, why is additional proteolytic cleavage of the p51↓RNH junction needed in vivo during virus maturation? We recently showed that mutagenesis of the RT p51↓RNH protease recognition sequence (AETF440↓ Y441VDG) resulted in aberrant proteolytic processing producing HIV-1 virions with greatly decreased levels of RT that in many cases was primarily RT p51, leading to substantially reduced replication capacity [22]
We propose that when the p51↓RNH junction is mutated, the T477A compensatory substitution may enable HIV-1 PR-mediated proteolytic processing of RT p66 at another site close to the normal proteolytic cleavage point, thereby enabling formation of an RT heterodimer refractory to additional proteolytic degradation

Summary

Introduction

The p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. KDa (p66) and 51 kDa (p51) subunits [1] The latter subunit, p51, is derived from the larger p66 subunit (or a larger RT precursor) by HIV-1 protease (PR)-catalyzed cleavage of the p51↓RNH junction during viral maturation. This event results in the removal of a 15 kDa Cterminal ribonuclease H (RNH) domain [2,3,4,5]. In addition to its catalytic function, the RNH domain of the p66 subunit has been suggested to play a structural role in the maintenance of RT stability [11,12,13,14,15,16]

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