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

Abstract

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particleassociated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Prototype Foamy Virus Gag Nuclear Localization: a Novel Pathway among Retroviruses

“…Cell culture supernatants containing recombinant viral particles were generated by transfection of 293T cells using polyethyleneimine (PEI) or the PolyFect transfection reagent as described previously (26,28,33). For subsequent Western blot analysis, the supernatant generated by transient transfection was harvested, passed through a 0.45-m filter, and centrifuged at 4°C and 25,000 rpm for 3 h in an SW40 or SW28 rotor (Beckman) through a 20% sucrose cushion.…”

“…A 4-component PFV vector system consisting of the expression vectors pcoPG4 (PFV Gag), pcoPE (PFV Env), pcoPP or pcziPol, (PFV Pol), and the enhanced green fluorescent protein (eGFP)-expressing PFV transfer vector pMD9 has been described previously (20,33,50). Due to biosafety issues, a previously described variant PFV Pol expression construct with catalytically inactive reverse transcriptase, pcoPP2 (Pol iRT) (32,33), was used for live-cell imaging experiments employing the 4-component PFV vector system. For microscopy analysis, the ␤-galactosidase (LacZ)-expressing PFV transfer vector pMD11 (20) was used instead of eGFP-expressing pMD9.…”

“…3A and 6A. Chemically synthesized oligonucleotides, coding for the peptide sequence of a duplicated simian vacuolating virus 40 (SV40)-NLS separated by a single glycine residue, were introduced in the previously described Gag expression vectors pcoPG4 (wt), pcoPG4 CeYFP (CeYFP wt) (50), pcoPG4 ⌬GRII (⌬GRII), and pcoPG4 ⌬GRII CeYFP (CeYFP ⌬GRII) (33). Together with a flexible glycine-serine linker, the SV40-NLS peptide was added to the C terminus of the respective Gag proteins to obtain the pcoPG4 SV40-NLS (wt SV40-NLS 1), pcoPG4 ⌬GRII SV40-NLS (⌬GRII SV40-NLS 1), pcoPG4 CeYFP SV40-NLS (wt CeYFP SV40-NLS 1), and pcoPG4 ⌬GRII CeYFP SV40-NLS (⌬GRII CeYFP SV40-NLS 1) constructs.…”

“…PFV Gag mutants that lack GRII and, therefore, nuclear localization are also characterized by reduced infectivity and defects in Pol packaging (33,45,52,57). To examine whether nuclear targeting of PFV Gag by an SV40-NLS can rescue these defects or whether Gag nuclear targeting by an alternative import mechanism rather impairs infectivity, viral particles were produced from 293T cells transfected with expression vectors for the Gag wt, Gag ⌬GRII, or the Gag SV40-NLS mutants, together with Env (pcoPE), Pol (pcziPol), and a transfer vector (pMD9).…”

Knockdown of the host cellular protein transportin 3 attenuates prototype foamy virus infection

“…The underlying mechanisms of the gradually increased PFV Gag nuclear accumulation have been pinpointed to glycinearginine-rich box II (GRII). 35) It harbors in its C-terminal part, an NLS that is responsible for the transport of nascent Gag into the nucleus. 34) To obtain additional evidence, the subcellular localization of PFV Gag in infected TNPO3 KD and control cells was further analyzed by an immunostaining assay as described above for PFV IN localization.…”