Abstract
BackgroundFoamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation.ResultsSeveral Prototype FV (PFV) Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85PR-RT and p40IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71Gag resulted in a significant copackaging of these proteins.ConclusionsNon-particle associated PFV Pol appears to be naturally released from infected cells by a yet unknown mechanism. The absence of particle-associated Pol precursor suggests its rapid processing upon particle incorporation. Analysis of different PFV Gag-Pol fusion constructs demonstrates that orthoretroviral-like Pol expression is compatible with FV replication in principal as long as fusion protein processing is possible. Furthermore, unlike orthoretroviruses, PFV particle release and infectivity tolerate larger differences in relative cellular Gag/Pol levels.
Highlights
Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein
A major difference was the absence of Pol cleavage products p85PR-RT and p40IN in supernatant pellets when Pol was expressed alone, whereas both processing products were present in the corresponding cell lysates (Figure 1A, lane 8, 14). This extracellular Pol precursor appeared to be present as free protein and not in a lipid membrane enveloped vesicular form because it was completely sensitive to subtilisin digestion (Figure 1A, lane 7, 8). This suggested that the Prototype FV (PFV) Pol precursor protein, but not its processing products, is released into the supernatant by non-conventional secretion mechanisms as it lacks a classical signal peptide sequence [19]
Pol precursor protein is frequently detected in PFV particle preparations of different origin [13,16,20,21]
Summary
Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. Spuma- or foamy viruses (FVs) are a special type of retroviruses that have adopted features in their replication strategy commonly found in both orthoretrovirinae and hepadnaviridae [reviewed in [1]]. In respect to their expression strategy for the overlapping viral capsid (Gag) and polymerase (Pol) open reading frames (ORFs), FVs. Orthoretroviruses express Pol exclusively as Gag-Pol fusion proteins from their full-length genomic RNA by ribosomal frameshift or termination read-through mechanisms [reviewed in [2]]. The Gag-Pol precursor itself is unable to correctly assemble into infectious orthoretroviral particles
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