The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions

Abstract

Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.Foamy viruses (FV) are complex retroviruses that comprise one of two subfamilies. The FV replication strategy differs in many ways from that of orthoretroviruses such as human immunodeficiency virus (HIV-1) and is similar in some respects to that of hepadnaviruses such as human hepatitis B viruses (HBV). One of the major differences between FV and orthoretroviruses is the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein. About one Gag-Pol is translated for every 20 Gag proteins, and such translational regulation balances the required levels of structural proteins and enzymatic proteins. The Gag-Pol fusion is packaged into virions by coassembly with self-assembling Gag proteins (reviewed in reference 9). However, FV Pol is synthesized from a separate spliced mRNA independently of Gag. This raises fundamental questions about how Pol expression is regulated and how Pol is selectively incorporated into assembling virus particles without Gag determinants. Recently, we have shown that Pol expression is regulated at the level of transcription by utilization of a suboptimal splicing site (14). The details of FV Pol packaging are not yet known. HBV also expresses its reverse transcriptase (RT) (P protein) independently of the structural proteins. However, HBV P protein is responsible for packaging of genomic RNA and initiating assembly of capsids (18), whereas FV Pol is not required for particle assembly or encapsdation of genomic RNA (1). Thus, the mechanism of FV Pol incorporation is unique, differing from those of orthoretroviruses and hepadnaviruses.

Novel Functions of Prototype Foamy Virus Gag Glycine- Arginine-Rich Boxes in Reverse Transcription and Particle Morphogenesis

“…However, it appears to be linked to the packaging of another essential component of the infectious viral particle, the viral genomic RNA (29,32). Current data suggest that protein-RNA interaction of both Gag and Pol with viral genomic RNA as well as protein-protein interactions between Gag and Pol are involved in the assembly process (14,21,29).…”

“…However, Pol expression is not fully Gag independent as mutations introduced at the C terminus of Gag influence the levels of spliced pol mRNA (21). As a consequence of separate Pol expression, FVs require a strategy different from that of orthoretroviruses to ensure Pol particle incorporation.…”

“…It is speculated that FV Gag, Pol, and the viral RNA form a ternary complex (21) in which all three components have distinct and equally important roles in a number of essential steps including Pol encapsidation, specific RNA genome recognition, RNA packaging, correct viral assembly, and reverse transcription. Processes such as the initial complex formation for particle assembly, Pol dimerization (12), protease activation, and Gag as well as Pol processing are also likely to be linked to these concerted actions.…”

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

“…Furthermore, it is assumed that the vRNA serves as a bridging molecule between Gag and Pol during assembly. There are conflicting results related to the requirement of additional protein-protein interactions between both proteins for PFV Pol incorporation (11)(12)(13). Interestingly, only the unprocessed Pol precursor protein appears to be efficiently packaged into assembling PFV particles (9,14).…”

Progress and prospects: Foamy virus vectors enter a new age

“…Recently, however, it was shown that Gag, too, contains C-terminal determinants that facilitate encapsidation of Pol into the virions. 12 This independent expression of Pol from a spliced mRNA may be an advantage for vector development, as a vector produced from three separate plasmids encoding each of the structural genes is less likely to undergo recombination. Foamy virus capsids are restricted to the cytoplasm in the absence of its cognate envelope protein and, thus, particles are not released into the supernatant in the absence of it or in the provision of a different envelope.…”