Foamy Retrovirus Integrase Contains a Pol Dimerization Domain Required for Protease Activation

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.

Section: Discussioncontrasting

confidence: 34%

contrasting

confidence: 46%

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

Hartl¹,

Bodem²,

Jochheim³

et al. 2011

“…Both cleavages can occur when PR is expressed as part of a Gag-Pol protein, consistent with results published by Löchelt et al (19). Like orthoretroviral PRs, PFV PR is active only as a homodimer (11,18,37). This implies that PFV Pol-Pol interactions can occur within the context of the Gag-Pol protein and that these interactions give rise to protease activity.…”

Section: Discussionmentioning

confidence: 99%

Foamy Virus Pol Protein Expressed as a Gag-Pol Fusion Retains Enzymatic Activities, Allowing for Infectious Virus Production

Lee

Sinicrope

Jackson

et al. 2012

Self Cite

Foamy viruses (FV) synthesize Pol from a spliced pol mRNA independently of Gag, unlike orthoretroviruses, which synthesize Pol as a Gag-Pol protein that coassembles with Gag. We found that prototype FV (PFV) mutants expressing Gag and Pol only as a Gag-Pol protein without the spliced Pol contain protease activity equivalent to that of wild-type (WT) Pol. Regardless of the presence or absence of the spliced Pol, the PFV Gag-Pol proteins can assemble into virus-like particles (VLPs), in contrast to the orthoretroviral Gag-Pol proteins, which cannot form VLPs. However, the PFV Gag-Pol VLPs have aberrant morphologies and are not infectious. In the absence of the spliced Pol, coexpression of a PFV Gag-Pol protein with Gag can produce infectious virions. Our results suggest that enzymes encoded by PFV pol (protease, reverse transcriptase, and integrase) are enzymatically active if they are synthesized as part of a Gag-Pol protein.

“…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). 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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Hütter

Müllers

Stanke

et al. 2013

bFoamy viruses (FVs) are unique among retroviruses in performing genome reverse transcription (RTr) late in replication, resulting in an infectious DNA genome, and also in their unusual Pol biosynthesis and encapsidation strategy. In addition, FVs display only very limited Gag and Pol processing by the viral protease (PR) during particle morphogenesis and disassembly, both thought to be crucial for viral infectivity. Here, we report the generation of functional prototype FV (PFV) particles from mature or partially processed viral capsid and enzymatic proteins with infectivity levels of up to 20% of the wild type. Analysis of protein and nucleic acid composition, as well as infectivity, of virions generated from different Gag and Pol combinations (including both expression-optimized and authentic PFV open reading frames [ORFs]) revealed that precursor processing of Gag, but not Pol, during particle assembly is essential for production of infectious virions. Surprisingly, when processed Gag (instead of Gag precursor) was provided together with PR-deficient Pol precursor during virus production, infectious, viral DNA-containing particles were obtained, even when different vector or proviral expression systems were used. Although virion infectivity was reduced to 0.5 to 2% relative to that of the respective parental constructs, this finding overturns the current dogma in the FV literature that viral PR activity is absolutely essential at some point during target cell entry. Furthermore, it demonstrates that viral PR-mediated Gag precursor processing during particle assembly initiates intraparticle RTr. Finally, it shows that reverse transcriptase (RT) and integrase are enzymatically active in the Pol precursor within the viral capsid, thus enabling productive host cell infection.