The RNA Binding G-patch Domain in Retroviral Protease Is Important for Infectivity and D-type Morphogenesis of Mason-Pfizer Monkey Virus

Jitka Stokrová,Eric Hunter,Tomás Ruml,Kvido Strísovský,Iva Pichová,Helena Bauerová-Zábranská

doi:10.1074/jbc.m508031200

Abstract

Retroviral proteases (PRs) cleave the viral polyprotein precursors into functional mature proteins late during particle release and are essential for viral replication. Unlike most retroviruses, beta-retroviruses, including Mason-Pfizer monkey virus (M-PMV), assemble immature capsids within the cytoplasm of the cell. The activation of beta-retroviral proteases must be highly regulated, because processing of the Gag-related polyprotein precursors occurs only after transport of immature capsids to the plasma membrane and budding. Several beta-retroviral proteases have unique C-terminal extension sequences, containing a glycine-rich motif (G-patch), which specifically binds in vitro to single-stranded nucleic acids. In M-PMV PR the G-patch is removed in vitro as well as in vivo by autoproteolytic processing to yield truncated active forms of PR. To investigate the role of the G-patch domain on the virus life cycle, we introduced mutations within the C-terminal domain of protease. We found that the G-patch domain of M-PMV PR is not required for the processing of viral polyproteins, but it significantly influences the infectivity of M-PMV, the activity of reverse transcriptase, and assembly of immature capsid within the cells. These results demonstrate for the first time that the G-patch domain of M-PMV PR is critical for the life cycle of beta-retroviruses, and its evolutionary conservation within members of this genus suggests its importance for retroviruses that display D-type morphology.

Highlights

The maturation process of retroviruses is mediated by a virus-encoded protease (PR)3 that functions to cleave the viral polyproteins into the mature structural proteins and enzymes found in infectious particles
These results demonstrate for the first time that the G-patch domain of Mason-Pfizer monkey virus (M-PMV) PR is critical for the life cycle of ␤-retroviruses, and its evolutionary conservation within members of this genus suggests its importance for retroviruses that display D-type morphology
In vitro experiments confirmed that the first autocatalytic processing of 17PR occurs at the C terminus in position Ala114Gln115 and it is followed by cleavage in position Ser107-Pro108 yielding the 13 and 12 kDa forms of M-PMV PR, respectively

Summary

Introduction

The maturation process of retroviruses is mediated by a virus-encoded protease (PR) that functions to cleave the viral polyproteins into the mature structural proteins and enzymes found in infectious particles. The dominant signal responsible for this targeting was identified within the N terminus of the Gag polyprotein in the matrix domain (MA) and has been termed the cytoplasmic targeting/retention signal (CTRS) [3, 4] This motif of 18-amino-acid spanning residues 43– 60 within MA sequence mediates intracytoplasmic targeting by interaction with the dynein/dynactin molecular motor complex [2]. The first proteolytically active M-PMV PR excised from Gag-precursors is a 17-kDa protein (per monomer) (17PR), which undergoes further C-terminal self-processing yielding 13-kDa protease (13PR). These two forms of protease were detected in released virions. A similar domain was identified in over 100 eukaryotic proteins many of which are involved in RNA processing [11]

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Conclusion