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

Abstract

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.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

“…Subsequently, FV Env leader peptide binds Gag to facilitate membrane targeting and particle release [45]. However, it has been demonstrated that cleavage of PFV p71-Gag to generate p68-Gag is required for the initiation of reverse transcription [66]. Furthermore, it has been shown that proteolytic processing of the Gag protein of S .…”

“…Cell culture supernatants containing recombinant viral particles were generated by transfection of the corresponding plasmids into 293T cells using polyethyleneimine (PEI) as described previously [66, 96]. For subsequent Western blot analysis the supernatant generated by transient transfection was harvested, passed through a 0.45-μm filter and centrifuged at 4°C and 25,000 rpm for 3 h in a SW32Ti rotor (Beckman) through a 20% sucrose cushion.…”

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

“…In some experiments previously described variants of the PFV Pol packaging construct, containing ORFs with catalytically inactive reverse transcriptase (pcoPP2, Pol iRT, YVDD 312–315 GAAA mutation) or catalytically inactive integrase (pcoPP3, Pol iIN, D 936 A mutation), were used [66, 67]. …”

Foamy Virus Assembly with Emphasis on Pol Encapsidation

“…In this experiment, the Gag-PR-RT fusion protein is incorporated into virions and exhibits PR activity in the absence of the IN domain, indicating that Gag can provide PR activation normally supplied by IN. A recent report showed that uncleaved PrPol is more efficient in Gag processing than the PR-RT cleaved subunit [57], supporting a role for IN in PR in wild-type infection. Characterization of the PR activation mechanism requires further investigation.…”

“…A recent study using a FV four-vector system found that Gag processing is required for initiation of reverse transcription [57]. It is not yet clear whether the precursor Gag protein somehow inhibits RT activity during assembly or whether the cleaved p3 peptide has a stimulatory effect on RT.…”