Regulation of Foamy Virus Protease Activity by Viral RNA: a Novel and Unique Mechanism among Retroviruses

Abstract

Foamy viruses (FVs) synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to what was found for orthoretroviruses, e.g., human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol consists of a protease (PR) domain, a reverse transcriptase domain, and an integrase domain and is processed into a mature protease-reverse transcriptase (PR-RT) fusion protein and the integrase. Protease activity has to be strictly regulated in order to avoid premature Gag and Pol processing before virus assembly. We have demonstrated recently that FV protease is an inactive monomer with a very weak dimerization tendency and postulated protease activation through dimerization. Here, we identify a specific protease-activating RNA motif (PARM) located in the pol region of viral RNA which stimulates PR activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that at least two foamy virus PR-RT molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2 hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation.

Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

“…In contrast to the human immunodeficiency virus type 1 (HIV-1) RT heterodimer (p66/ p51), prototype FV (PFV) PR-RT has been shown to be monomeric and able to catalyze all RT-related functions (3), while PR activation requires dimer formation of the PR domain via the binding of PR-RT to a specific element on the viral RNA, called the protease-activating RNA motif (PARM) (4,5). Hence, for the retrotranscription functions, PFV PR-RT differs from alpharetroviral and lentiviral RTs (including HIV-1 RT), which are dimeric, and it is similar to the gammaretroviral RTs that are monomeric (6).…”

Prototype Foamy Virus Protease Activity Is Essential for Intraparticle Reverse Transcription Initiation but Not Absolutely Required for Uncoating upon Host Cell Entry

“…Whereas Lee et al (19) reported a role of the IN domain within the precursor protein for inducing PR dimerization and enzymatic activity, Hartl and colleagues (18) described the stimulation of PFV PR activity by a PFV vRNA element, the protease-activating RNA motif (PARM). PFV PR dimerization by PARM involves two purine-rich sequences and seems to be mediated by RNA binding to the RT domain with no need for the IN domain (18,20).…”

“…However, mature PFV p85 PR-RT was shown to be monomeric in solution and to lack proteolytic activity under physiologically relevant conditions in vitro (17). The mechanism of PR activation is a controversial subject (18,19). Whereas Lee et al (19) reported a role of the IN domain within the precursor protein for inducing PR dimerization and enzymatic activity, Hartl and colleagues (18) described the stimulation of PFV PR activity by a PFV vRNA element, the protease-activating RNA motif (PARM).…”

Foamy Virus Gag p71-p68 Cleavage Is Required for Template Switch of the Reverse Transcriptase

“…We propose that by this mechanism cDNA synthesis is delayed until sufficient amounts of Gag and Pol are cleaved to ensure viral infectivity. This checkpoint is needed, because cDNA synthesis leads to degradation of the protease-activating RNA motif (PARM), which is essential for protease activity in FV (8) and therefore terminates processing of Gag and Pol.…”

“…First, we exchanged the wildtype Gag cleavage site sequence RAVN-TVTQ with residues GAL G-ALGA in the context of the codon-optimized expression plasmid ( Fig. 1) (8,9). This plasmid was named p71⌬CS (Fig.…”