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, particle-associated 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.

The Retroviridae consist of two subfamilies, the Orthoretrovirinae with six different genera and the Spumaretrovirinae with only one genus, i.e., that of the spumaviruses, which are also known as foamy viruses (FVs). A thorough analysis of FVs has revealed some remarkable differences in their replication strategy compared to that of orthoretroviruses (reviewed in reference 32). Indeed, some features are strikingly similar to those of another family of reverse transcriptase (RT)-encoding viruses, the Hepadnaviridae (2).

One of these crucial differences in FV replication strategy is the expression of Pol from a separate pol mRNA rather than expression from a gag-pol mRNA. 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. The actual details of this mechanism are still largely unknown. 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).

Another distinguishing feature is the time point of reverse transcription. In contrast to orthoretroviruses, FVs can reverse transcribe their genome late in the viral life cycle. Although an early reverse transcription step has been described (5, 46), reverse transcription largely occurs in the virus-producing cell during or immediately after capsid assembly and prior to virus budding. Furthermore, DNA-containing prototype foamy virus (PFV) particles represent the major form of infectious particles (27, 33, 45). Proposed mechanisms of FV capsid assembly, encapsidation of Pol, and RNA packaging must therefore also take into account reverse transcription.

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.

The exact roles of each of the three players—Gag, Pol, and genomic RNA—in these crucial and complex processes are not completely understood for FVs, nor is much known about the possible contributions of cellular proteins. However, these three components and their respective contributing domains have been well studied in orthoretroviruses. Orthoretroviral Gag is processed by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) subunits. Of these major domains, MA and particularly NC have been shown to play a significant role in the encapsidation of the viral RNA (reviewed in reference 25). The NC subunit, which can be found in all orthoretroviruses, contains a high number of basic residues and, most notably, one or two copies of a conserved motif composed of regularly spaced cysteine and histidine residues, the Cys-His motif. The Cys-His motifs from different orthoretroviruses are known to be involved in viral genomic RNA recognition and packaging, particle assembly, and reverse transcription (reviewed in reference 18).

In contrast to orthoretroviruses, FV replication strategy differs with respect to its capsid protein biogenesis. In FV morphogenesis there is no processing of Gag into MA, CA, and NC but, rather, only limited proteolysis, resulting in the removal of a small C-terminal peptide. Released infectious PFV particles are composed of both the uncleaved precursor p71^Gag and the larger p68^Gag cleavage products in ratios of 1:1 to 1:4 (4). Despite the lack of extensive precursor processing, FV MA, CA, and NC domains were originally assigned to the N terminus, the central region, and the C terminus of FV Gag, respectively (10, 44). However, the FV Gag “NC” domain lacks the Cys-His motifs found in orthoretroviral Gag. Instead, a large percentage of glycine (G) and arginine (R) residues are found in the C-terminal region of Gag for all FV species (25, 43). In simian FVs (SFV) and PFV Gag, these are clustered in three GR-rich boxes (GR boxes) (36, 40). For the Gag proteins of nonprimate FVs, feline FV (FFV), bovine FV (BFV), and equine FV (EFV), no clustering of GR residues is observed, and only small peptide motifs of GR box II (GRII) and GR box III (GRIII) are conserved throughout all FV Gag proteins (25, 35, 42, 43). The GR-rich FV Gag C terminus and, in particular, the clustered GR boxes of primate FVs are thought to play roles in viral replication similar to those of the Cys-His motifs of orthoretroviruses (32).

However, present experimental evidence gives only sparse support for such functions. In particular, the PFV GR box I (GRI) has been implicated in RNA as well as in Pol packaging. In an early study, GRI was found to bind nucleic acids in vitro, and it was therefore originally implicated in RNA binding (44). Another study achieved similar results by stop codon introduction in front of each GR box in the proviral context (38). This study also suggested that Pol is packaged in the absence of GRIII, whereas GRI and GRII are required. Infectivity was decreased by 2 orders of magnitude in truncation mutants lacking the C-terminal 3-kDa peptide and was below the detection limit for truncations that deleted GRIII. Furthermore, these truncation mutants showed decreased Pol expression due to reduced levels of spliced pol mRNA and defects in Pol cleavage (21, 38). These and other studies strengthened the view that in PFVs, Gag, via GRI, and Pol precursor proteins bind to (pre)genomic RNA, where the RNA serves as a bridging molecule (13, 29, 38, 44). However, this conclusion was recently challenged by the finding that a GRI deletion mutant packages RNA in amounts similar to those of the wild-type (wt) virus (21). As a result, another function as a Gag-Pol protein interaction motif was proposed for GRI.

PFV GRII was reported to harbor in its C-terminal region a nuclear localization signal (NLS) responsible for transient nuclear targeting of PFV Gag at certain time points of the viral replication process (36, 44). Mutants of PFV GRII lack nuclear localization but still replicate in vitro (36, 44). In addition, a chromatin binding site (CBS) mediating attachment of PFV Gag to host chromosomes was identified in the N-terminal region of GRII (41). A mutant harboring point mutations in the CBS, analyzed in a four-plasmid vector system, retained about 15% infectivity compared to that of the wild type. Although GRII is best conserved between all FV species, only primate FV and EFV Gag proteins were reported to localize to the nucleus while FFV Gag does not (1, 20, 36). The reason for this discrepancy remains to be elucidated.

A study by Catellieri et al. focusing on the Gag domains required for PFV particle assembly and export also partly addressed the C terminus of Gag harboring the GR boxes (3). The authors used a four-plasmid vector system in which all components (Gag, Pol, Env, and genomic RNA) are expressed independently. In contrast to the situation with the provirus, the introduction of mutations in the structural proteins in this system did not influence the expression levels of other viral proteins, and there were no negative effects resulting from alterations in potential secondary structures of the (pre)genomic RNA to be incorporated into the virion. However, the conclusions of this study with regard to PFV GR box functions are limited because only C-terminal Gag truncations were used, and analysis was restricted to particle release and infectivity. Particle release was found to be unaffected, even for truncations comprising all GR boxes. However, the infectivity analyses with respect to GR box contributions remained inconclusive because, as reported previously, truncation of the C-terminal 3-kDa peptide was already sufficient to severely reduce viral titers (3, 47). Truncations resulting in the removal of GRII and GRIII (or more) completely abrogated infectivity.

There are, therefore, hints that the three GR boxes in the C terminus of PFV Gag are involved in the processes of genome recognition as well as RNA and Pol packaging. However, the data generated so far were almost exclusively obtained using proviral mutants, and therefore secondary effects contributing to the observed phenotypes cannot be excluded (21, 38, 44). Furthermore, many of the previous studies are inconclusive or fragmentary because they fail to investigate all three GR boxes in a comparable manner (4, 21, 38, 44).

In this study we provide the first simultaneous analysis of all three PFV Gag GR boxes in a directly comparable manner using a four-plasmid vector system. By detailed analysis of particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization, we present for the first time evidence for functions in reverse transcription and viral assembly. Furthermore, this analysis suggests that PFV Gag GR boxes are indeed functionally similar to orthoretroviral Cys-His motifs.

MATERIALS AND METHODS

Cells and culture conditions.

The human kidney cell line 293T (9), the human fibrosarcoma cell line HT1080 (30), and the human epithelium HeLa cell line (11) were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics. HeLa cells used for microscopy analysis were cultivated in phenol red-free medium.

Recombinant plasmid DNAs.

The original four-plasmid PFV vector system consisting of pcziPG4 (PFV Gag), pcziPol (PFV Pol), pczHFVenvEM002 (PFV Env), and the enhanced green fluorescent protein (eGFP)-expressing PFV transfer vector pMD9 has been described previously (13). In our study the expression-optimized constructs pcoPG4 (PFV Gag) (39), pcoPE (PFV Env), and pcoPP (PFV Pol) were used in place of the original Gag-, Pol-, and Env-expressing constructs. For some experiments, the variant PFV Pol expression construct pcoPP2 (with an inactive RT domain [iRT]), encoding an expression-optimized reverse transcriptase Pol protein catalytically inactivated through the M69 (YVDD_312-315GAAA) mutation described previously (27), was used. Expression optimization and gene synthesis were performed by Geneart, Germany. The individual PFV Gag constructs used in this study are depicted in Fig. 1A and B and 4A and B. Expression vectors for the Gag constructs are based on the previously described pcoPG4 and pcoPG4 CeYFP (where CeYFP is a C-terminal enhanced yellow fluorescent protein) vectors (39). GR box deletion constructs were generated by PCR amplification of two fragments. For both fragments one primer amplified a unique cloning site in the Gag expression construct (Eco91I upstream or NotI downstream from the GR box region) while the second primer annealed directly outside the respective GR box sequence introducing a unique BshTI and NheI restriction site through its 3′ end. Subsequently both PCR fragments were ligated into the pcoPG4 or pcoPG4 CeYFP vector using the Eco91I and the NotI restriction site of the vector constructs and the BshTI restriction site of the amplified fragments to fuse the fragments. As a result, 26 amino acids (aa) were deleted from each GR box, and a unique BshTI as well as a NheI restriction site encoding four new amino acids—T, G, A, and S—was introduced. Substitution and replacement constructs were generated by annealing chemically synthesized sense and antisense oligonucleotides, overlapping for the BshTI and NheI restriction sequence, and coding for the respective peptide sequence. Annealed oligonucleotides were ligated into the unique BshTI and NheI restriction sites of the respective GR box deletion construct. All constructs were verified by sequencing analysis. Primer sequences and additional details are available upon request.

FIG. 1.

Cellular and particle-associated protein expression analysis of the PFV Gag GR box deletion and substitution constructs. (A) Schematic outline of the generated PFV Gag GR box deletion and substitution constructs in wild type (PG) and C-terminal eYFP fusion (PG CeYFP) contexts. (B) PFV Gag GR box mutants were generated as deletions (ΔGRI, ΔGRII, and ΔGRIII) or alanine substitutions (GRI-Ala, GRII-Ala, and GRIII-Ala). The respective amino acid sequences are indicated. Numbers indicated the amino acid position in Gag. Artificially introduced amino acids are indicated in italics. The boxed amino acids indicate the wild-type sequence of each GR box as originally designated by Schliephake et al. (36). CMV, cytomegalovirus virus promoter; SD, splice donor; SA, splice acceptor; pA, bovine growth hormone polyadenylation signal; eYFP, enhanced yellow fluorescent protein. The p68/p71 PFV Gag cleavage site is shown as a dashed line. (C and D) Representative Western blot analysis of 293T cell lysates (cell) (C) and viral particles (virus) purified by ultracentrifugation through 20% sucrose for Gag GR box mutants (D). PFV proteins were detected by using specific antibodies for PFV Gag (α-Gag), PFV Pol PR/RT (α-PR/RT), PFV Pol IN (α-IN), or PFV Env LP (α-LP). 293T cells were cotransfected with pMD9, pcoPP, pcoPE, and either pcoPG4 (wt), pcoPG4 ΔGRI (ΔGRI), pcoPG4 ΔGRII (ΔGRII), pcoPG4 ΔGRIII (ΔGRIII), pcoPG4 GRI-Ala (GRI-Ala), pcoPG4 GRII-Ala (GRII-Ala), and pcoPG4 GRIII-Ala (GRIII-Ala). As controls, cells were transfected with pMD9, pcoPE, pcoPG4, and pcoPP2 (iRT); with pMD9, pcoPG4, and pcoPP (ΔEnv); or with only pcDNA3.1 zeo+ (mock). (E) Relative amounts of released Gag and RT quantified from Western blots from four independent experiments. In each experiment the values obtained in the ΔEnv lane were subtracted as background signal from the individual samples. Sample values were normalized for intracellular expression levels of Gag or Pol. The values obtained using wild-type PFV Gag expression plasmids were arbitrarily set to 100%. Data are the means ± standard deviations. Differences between means of wt and the individual mutants were analyzed by Welch's t test (***, P < 0.01).

Virus production and infectivity analysis.

Cell culture supernatants containing recombinant viral particles were generated as described previously (22, 24). Briefly, 293T cells were cotransfected with a Gag expression plasmid (pcoPG4 or PG mutants thereof, as indicated on the figures and in the figure legends), Env (pcoPE), Pol (pcoPP), and the transfer vector (pMD9) at a ratio of 4:1:2:28 using polyethyleneimine (PEI) or Polyfect transfection reagent. At 24 h posttransfection (p.t.), sodium butyrate (final concentration, 10 mM) was added to the growth medium for 8 h. The supernatant was harvested 48 h following transfection.

Analysis of the transduction efficiency of recombinant eGFP-expressing PFV vector particles was performed by infection of 2 × 10⁴ HT1080 cells, plated 24 h in advance in 12-well plates. One milliliter of the viral supernatant or serial dilutions thereof was incubated with the target cells. The percentage of eGFP-expressing cells was determined by fluorescence-activated cell sorter (FACS) analysis 72 h after infection. All transduction experiments were performed at least three times. In each independent experiment, the values obtained with the wild-type construct pcoPG4 were arbitrarily set to 100%, and values obtained with other than wild-type constructs were normalized as percentages of the wild-type values.

Purification of particulate material by ultracentrifugation.

Supernatant generated by transient transfection (as described above) was harvested, passed through a 0.45-μm-pore-size filter, and centrifuged at 4°C and 25,000 rpm for 3 h in a SW40 or SW28 rotor (Beckman) through a 20% sucrose cushion. The particulate material was resuspended in phosphate-buffered saline (PBS).

Immunoblotting and antisera.

Cellular protein lysates from one transfected 10-cm cell culture dish were prepared by a 20-min incubation with 0.6 ml of detergent-containing buffer on ice and subsequent centrifugation of the lysates through a QIAshredder (Qiagen). Protein samples from cellular lysates or purified particulate material were separated by SDS-PAGE using a 10% polyacrylamide gel and analyzed by immunoblotting as described previously (23). Polyclonal rabbit antiserum specific for PFV Gag (37) or amino acids (aa) 1 to 86 of the PFV Env leader peptide (LP), (23) as well as a hybridoma supernatant specific for PFV protease/reverse transcriptase (PR/RT) (clone 15E10) or PFV integrase (IN) (clone 3E11) (17), was used. After incubation with a suitable horseradish peroxidase (HRP)-conjugated secondary antibody, the blots were developed with Immobilon Western HRP substrate (Millipore). The chemiluminescence signal was digitally recorded using an LAS-3000 imager. Western blot signal intensities were quantified using ImageJ software.

qPCR analysis.

Sample processing for quantitative PCR (qPCR) was essentially conducted as described previously (26). The qPCRs were carried out using 5 μl of each reverse transcription reaction mixture analyzed in duplicates in a total volume of 25 μl using a Brilliant II QPCR Kit (Stratagene) and an MX4000 Multiplex Quantitative PCR System (Stratagene). Primers (sense, 5′-CTTCAACCTTTGCTGAATG-3′; antisense, 5′-TAATACAGGGCTATAGGTGT-3′) and TaqMan probe (6′-FAM-TTGGAATTCAGTACTCCTTATCACCC-3′-BHQ1, where FAM is 6-carboxyfluorescein and BHQ is Black Hole quencher) were specific for a conserved part of the residuary Pol region present in the cis-acting sequence II (CASII) element of the pMD9 vector construct and used at 5 pmol/μl. Samples were initially denatured for 8 min at 95°C and subsequently amplified in 50 cycles of 60 s at 95°C, 1 min at 58°C, and 15 s at 72°C. Amounts were determined in reference to a standard curve prepared by serial dilution of pMD9 plasmid. All qPCR experiments were performed at least three times. In each independent experiment the values obtained for the viral genome (DNA or RNA) with the wild-type construct pcoPG4 were arbitrarily set to 100%, and values obtained with other than wild-type constructs were normalized as percentages of the wild-type values.

Reverse transcriptase assay.

Particle-associated exogenous RT activity was analyzed as described previously (26).

Fluorescence microscopy analysis.

HeLa cells were seeded at a density of 6 × 10⁴ cells/well on coverslips into 12-well plates. After 24 h cells were transfected with a Gag C-terminal eYFP fusion expression plasmid (pcoPG4 CeYFP or mutants thereof, as indicated on the figures and in the figure legends), alone or together with Env (pcoPE), Pol (pcoPP), and the transfer vector (pMD11) at a ratio of 4:1:2:28 using FuGene transfection reagents. At 48 h p.t., cells were washed with cold PBS and fixed with 3% paraformaldehyde. After fixation the cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min. Finally, the cells were covered with Mowiol. Confocal laser scanning images were obtained on a Zeiss LSM 510 using a Zeiss Apochromat 63× (numerical aperture, 1.4) oil immersion objective using excitation light of an argon laser at 488 nm or a diode laser at 405 nm, reflected by a dichroic mirror (HFT 405/488/561). Fluorescence signal was recollected by the same objective, split by a dichroic mirror (NFT 490), passed through either a 520/30 or a 420/60 band-pass filter, and subsequently measured by a photomultiplier tube (PMT). Confocal geometry was ensured by a 1-Airy unit pinhole in front of the PMT. Fluorescence images were evaluated using ImageJ software.

Electron microscopy analysis.

At 48 h p.t., 293T cells transiently transfected with the individual PFV Gag expression constructs as indicated on the figures and in the figure legends in cotransfection with Env (pcoPE), Pol (pcoPP) and transfer vector (pMD9) were harvested and processed for electron microscopy analysis as described previously (19).

Statistical analysis.

Amounts of released Gag and RT were quantified from Western blots from four independent experiments and normalized to the intracellular expression (see Fig. 1E). Viral titers of the PFV Gag GR box mutants were determined in duplicates in four independent experiments (see Fig. 3). Viral titers of the PFV Gag GR box replacement constructs were determined in duplicates in three independent experiments (see Fig. 4). Particle-associated RNA and DNA amounts were determined in duplicates in three to four independent experiments and normalized to particle release (see Fig. 5A). Particle-associated exogenous reverse transcriptase activity was determined in duplicates in three to four independent experiments and normalized to particle release (see Fig. 5B). Under each experimental condition the values obtained using wild-type PFV Gag were arbitrarily set to 100%.

Results are presented as means ± standard deviations. Welsh's t test was used to evaluate differences between the mean of an individual mutant and the mean of the wt sample (28, 34).

RESULTS

Generation of PFV Gag GR box deletion and substitution.

PFV Gag contains three GR boxes with lengths of 28 aa (GRI), 23 aa (GRII), and 34 aa (GRIII) in its C terminus (Fig. 1A) (36). Previous investigations of their function were conducted almost exclusively in the proviral context (38, 44). Deletions or substitutions of the GR boxes examined in such studies comprised at most 11 aa or were generated as C-terminal truncation mutants by introduction of stop codons (3, 38, 44). To investigate all three GR boxes in a similar and comparable manner, equal size deletions were created in the previously described PFV Gag expression vectors pcoPG4 (PG wt) (Fig. 1A and B) (39). For analysis of intracellular distribution and trafficking, the mutants were examined in the context of the pcoPG4 CeYFP (PG CeYFP) expression vector encoding a C-terminal eYFP-tagged Gag fusion protein (Fig. 1A and B). In all GRIII box mutants the C-terminal 8 aa of the originally designated GRIII box sequence, containing one G and one R residue, were retained to avoid potential interference with the neighboring p68^Gag/p3^Gag cleavage site processing. The mutant Gag constructs were cotransfected with expression vectors for Env (pcoPE), Pol (pcoPP), and a transfer vector (pMD9) into 293T cells. Viral protein expression was examined in cell lysate samples using Western blotting. All Gag GR box mutants expressed and processed the predicted Gag protein at levels similar to the level of the wild type (Fig. 1C, lanes 2 to 7). However, GRII box mutants were recovered slightly better in the cell lysates. Cellular expression levels of PFV Pol and Env were not influenced by the individual Gag mutants (data not shown).

Particle release and particle protein composition.

One of the first steps of FV capsid assembly is the multimerization of Gag proteins in the cytoplasm. RNA and Pol are packaged into the assembling capsids. A specific interaction of FV Gag and Env is essential for the trafficking of the preassembled capsids to cellular membranes where viral particle release occurs. To determine whether the PFV Gag GR box deletion and substitution mutants retained the ability to assemble and release particles in wt-like amounts and with wt-like protein composition, we performed Western blotting with anti-Gag, anti-RT, anti-IN, or anti-Env antibodies on pelleted viral supernatants (Fig. 1D). To control for nonspecific release of viral proteins into the supernatant, pelleted supernatant from 293T cells transfected with Gag, Pol, and transfer vector but lacking Env (ΔEnv) was also examined (Fig. 1D, lane 9). No significant differences in the cellular Pol and Env expression levels of the individual mutant Gag samples in comparison to the wild-type control were observed (data not shown). Each of the Gag mutants released viral particles in apparently wt-like amounts with wt-like protein composition and processing (Fig. 1D, lanes 1 to 7). Normalized to cellular expression levels, the ΔGRII mutant showed a statistically significant, but only 2-fold reduced, amount of released Gag (Fig. 1E). However, this might be due to a better recovery of GRII box mutants in cell lysates as a result of altered intracellular localization (see below). Interestingly, despite identically sized deletions, Gag mutants with a deleted GRII migrated at a slightly higher molecular weight than the analogous GRI and GRIII deletion mutants (Fig. 1D, lanes 2 to 4). Furthermore, Pol was found in the viral supernatants for all mutants (Fig. 1D). Again, only ΔGRII showed statistically significant (3-fold) reduction of particle-associated RT when values were normalized for cellular expression levels (Fig. 1E). The RT and IN subunits detected in pelleted viral particles proved resistant to subtilisin digestion and therefore must be shielded by a lipid membrane (data not shown). The Pol precursor was also detected in the viral supernatants of all mutants and of the ΔEnv control. However, it was sensitive to subtilisin digestion and therefore not particle associated (A. Swiersy et al., submitted for publication). Pol and Gag cleavage was observed only for particle-associated Pol (Fig. 1D, lanes 1 to 8).

Therefore, deletion or substitution of any one of the three GR boxes does not substantially impair particle release, viral particle protein composition, or processing of viral proteins. Most notably, in contrast to the proviral context (21), in the four-plasmid vector system, Pol is packaged into viral particles in the absence of GRI.

Intracellular localization.

A long-recognized hallmark of primate FV infection is the transient nuclear localization of the Gag protein. Although its biological significance remains unclear, the underlying mechanisms were pinpointed to GRII, which harbors in its C-terminal part an NLS reported to be responsible for the transport of nascent Gag into the nucleus (36, 44) and in its N-terminal part a CBS which was proposed to tether incoming PFV preintegration complexes (PIC) onto host chromatin (41). We transfected into HeLa cells Gag GR box mutants with C-terminal fusions of eYFP solely or in combination with expression vectors for Env (pcoPE), Pol (pcoPP), and a transfer vector (pMD11) in order to determine their intracellular localization. Cells were fixed and processed for confocal imaging at 48 h p.t. Confocal microscopy analysis of Gag-only transfections revealed wt-like cellular localization for GRI and GRIII mutants with a homogenous nuclear signal and some accumulation of cytoplasmic Gag in close proximity to the nucleus, presumably the pericentriolar region (Fig. 2, wt, ΔGRI, ΔGRIII, GRI-Ala, and GRIII-Ala). The GRII deletion and substitution mutants displayed similar cytoplasmic localizations but completely lacked nuclear localization of Gag (Fig. 2, ΔGRII and GRII-Ala). Cotransfection of Gag with Pol, Env, and transfer vector did not alter Gag localization compared with that of Gag-only transfection (data not shown).

FIG. 2.

PFV Gag GR box mutant intracellular localization. HeLa cells were transfected with expression plasmids for C-terminal eYFP-tagged Gag GR box substitutions or deletions pcoPG4 CeYFP (wt), pcoPG4 CeYFP ΔGRI (ΔGRI), pcoPG4 CeYFP ΔGRII (ΔGRII), pcoPG4 CeYFP ΔGRIII (ΔGRIII), pcoPG4 CeYFP GRI-Ala (GRI-Ala), pcoPG4 CeYFP GRII-Ala (GRII-Ala), or pcoPG4 CeYFP GRIII-Ala (GRIII-Ala). At 48 h p.t. the samples were fixed, stained with DAPI, and covered in Mowiol. Columns labeled eYFP (Gag) and DAPI (DNA) (gray channel) show the signals obtained in the respective channels. The merge column shows a merged image of the eYFP (green) and the DAPI (red) channels. Scale bar, 10 μm.

Infectivity.

Previous studies analyzed GRI, GRII, and GRIII substitution mutants and GRI and GRII deletion mutants for infectivity in the proviral context (21, 44). The authors did not detect infectivity for mutants with GRI or GRII replaced by a hemagglutinin (HA) tag. In contrast, GRI and GRII deletion mutants as well as a mutant with GRIII replaced by an HA tag retained measurable infectivity. We determined infectivity of our GR box mutants by a marker gene transfer assay using a four-plasmid vector system. Our results showed approximately 2%, 5%, and 9% of wild-type infectivity remaining for deletion mutants ΔGRI, ΔGRII, and ΔGRIII, respectively (Fig. 3, ΔGRI, ΔGRII, and ΔGRIII). Surprisingly, none of the GR box alanine substitution (GR-Ala) mutants yielded a measurable titer (Fig. 3, GRI-Ala, GRII-Ala, and GRIII-Ala). This represents at least a 4-log reduction in titer compared to the wild type.

FIG. 3.

Infectivity analysis of the PFV Gag GR box deletion and substitution constructs. PFV particle-containing cell culture supernatants were generated by transient transfection of 293T cells using the four-plasmid PFV vector system. Relative infectivity of extracellular cell culture supernatants using an eGFP marker gene transfer assay were measured 3 days postinfection. 293T cells were cotransfected with pMD9, pcoPP, pcoPE, and either pcoPG4 (wt), pcoPG4 ΔGRI (ΔGRI), pcoPG4 ΔGRII (ΔGRII), pcoPG4 ΔGRIII (ΔGRIII), pcoPG4 GRI-Ala (GRI-Ala), pcoPG4 GRII-Ala (GRII-Ala), or pcoPG4 GRIII-Ala (GRIII-Ala). As control, cells were transfected with pcDNA3.1 zeo+ (mock) alone. The values obtained using wild-type PFV Gag expression plasmids were arbitrarily set to 100%. Means and standard deviations from four independent experiments are shown. Differences between means of wt and the individual mutants were analyzed by Welch's t test (***, P < 0.01).

Therefore, while the GR boxes are not essential for assembly and release of virus particles in wt-like amounts or for protein composition and protein processing, they are very important for viral infectivity. Unexpectedly, alanine substitutions seem to totally abolish infectivity.

Replacement and rescue of the PFV GR boxes.

We generated the deletion mutants by replacing the GR box DNA sequence with two unique restriction sites (BshTI and NheI) to allow for a subsequent modular domain exchange. These foreign sequences encode a TGAS peptide originally not present in PFV Gag (Fig. 1B). To test this modular system and to determine whether the four introduced amino acids themselves cause an altered phenotype, we inserted at each deletion site oligonucleotide linkers coding for the deleted original 26-aa GR box peptides (rescue mutants GRI-I, GRII-II, and GRIII-III). Furthermore, to examine whether the individual GR boxes can complement each other functionally, the amino acid sequences of the other two GR boxes were inserted into the individual GR box deletion mutants (replacement mutants GRI-II, GRI-III, GRII-I, GRII-III, GRIII-I, and GRIII-II) (Fig. 4A and B).

FIG. 4.

Infectivity analysis of PFV Gag GR box replacement constructs. PFV particle-containing cell culture supernatants were generated by transient transfection of 293T cells using a four-plasmid PFV vector system. (A) CMV, cytomegalovirus virus promoter; SD, splice donor; SA, splice acceptor; pA, bovine growth hormone polyadenylation signal; eYFP, enhanced yellow fluorescent protein. The p68/p71 PFV Gag cleavage site is shown as a dashed line. (B) Schematic outline of the generated PFV Gag GR box replacement constructs. PFV Gag GR box mutants were generated by introducing the respective wild-type GR box amino acid sequence in place of the various authentic GR box peptides. The amino acid sequences and positions are indicated. Artificially introduced amino acids are indicated in italics. The boxed amino acids indicate the wild-type sequence of each GR box as originally determined by Schliephake et al. (36). (C) Relative infectivity of extracellular cell culture supernatants using an eGFP marker gene transfer assay measured at 3 days postinfection. The values obtained using wild-type PFV Gag expression plasmids were arbitrarily set to 100%. Means and standard deviations from at least three independent experiments are shown. Differences between means of the wt and the individual mutants were analyzed by Welch's t test (***, P < 0.01). 293T cells were cotransfected with pMD9, pcoPP, pcoPE, and either pcoPG4 (wt), pcoPG4 ΔGRI (ΔGRI), pcoPG4 GRI-I (GRI-I), pcoPG4 GRI-II (GRI-II), pcoPG4 GRI-III (GRI-III), pcoPG4 ΔGRII (ΔGRII), pcoPG4 GRII-I (GRII-I), pcoPG4 GRII-II (GRII-II), pcoPG4 GRII-III (GRII-III), pcoPG4 ΔGRIII (ΔGRIII), pcoPG4 GRIII-I (GRIII-I), pcoPG4 GRIII-II (GRIII-II), or pcoPG4 GRIII-III (GRIII-III). As a control, cells were transfected with only pcDNA3.1 zeo+ (mock).

All rescue and replacement mutants together with the GR box deletion mutants were analyzed for infectivity relative to that of the wild type. By introduction of the original GR box sequence, infectivity was rescued to 106%, 115%, and 136% for mutants GRI-I, GRII-II, and GRIII-III (Fig. 4C). Therefore, the four artificially introduced amino acids do not have a negative effect.

The result of replacing each GR box with one of the other GR boxes was interesting. It has been reported that GRI can be replaced with GRII or GRIII with retention of 70 to 80% of wild-type infectivity (21). We found that, in terms of particle infectivity, the GRIII sequence functionally substitutes for GRI sequence more efficiently than does the GRII sequence and, vice versa, the GRI sequence functionally substitutes for GRIII sequence better than the GRII sequence (Fig. 4C). In line with this, functional complementation of GRII by GRI and GRIII sequences was poor (Fig. 4C). The data therefore imply that GRI and GRIII are more closely related to each other than to GRII.

Nucleic acid content and reverse transcription.

A tempting explanation for the severe decrease of infectivity in deletion mutants and a total lack of infectivity in substitution mutants is a failure in the encapsidation or reverse transcription of the viral genomic RNA. To determine whether these mutants package and subsequently reverse transcribe RNA, qPCR analysis for DNA content and reverse transcription-qPCR (RT-qPCR) analysis for RNA content were performed. All PFV Gag mutants packaged vector RNA at amounts approximately 50 to 70% of that of the wild type (Fig. 5A). Furthermore, DNA content was found to be about 1.2%, 5.2%, and 3.1% for PFV Gag GR box deletion mutants (ΔGRI, ΔGRII, and ΔGRIII) and below 0.1% for GR-Ala substitution mutants, a 3-log reduction (Fig. 5A). For the wild-type PFV Gag vector cotransfected with a Pol expression construct harboring an inactive RT domain (iRT), RNA amounts were determined to be approximately 150%, but DNA content was below 0.1% compared to that of the wild type. The measured DNA value of this sample reflects the detection limit of the RT-qPCR assay employed. Similar background signals for DNA and also RNA values were observed in supernatant samples of 293T cells transfected either with a combination of Gag, Pol, and transfer vector but omitting Env or solely with the pMD9 transfer vector (data not shown).

FIG. 5.

Nucleic acid composition and exogenous reverse transcriptase activity of mutant PFV particles. Mutant PFV particles as indicated were generated by transient transfection of 293T cells using a four-plasmid PFV vector system. (A) Relative nucleic acid composition of mutant particles as determined with PFV Pol-specific primers and TaqMan probe. Following DNase I digestion of intact, purified particles, nucleic acids were isolated, and the relative amount of vector RNA and number DNA copies were determined in comparison to levels in the wild type by qPCR. The mean values and standard deviations of the relative RNA and DNA content of at least three independent experiments are shown. Sample values are normalized for capsid protein release. Differences between means of the wt and the individual mutants were analyzed by Welch's t test (***, P < 0.01). (B) Relative exogenous reverse transcriptase activity of mutant particles. Following subtilisin digestion of intact, purified particles and a subsequent additional ultracentrifugation step, PFV exogenous reverse transcriptase activity was determined from particle lysates using a RetroSys C-type RT enzyme-linked immunosorbent assay. The mean values and standard deviations of the relative reverse transcriptase activity of at least three independent experiments normalized to the amount of RT detected in Western blotting of the individual mutants are shown. Differences between means of the wt and the individual mutants were analyzed by Welch's t test (***, P < 0.01). 293T cells were cotransfected with pMD9, pcoPP, pcoPE, and either pcoPG4 (wt), pcoPG4 ΔGRI (ΔGRI), pcoPG4 ΔGRII (ΔGRII), pcoPG4 ΔGRIII (ΔGRIII), pcoPG4 GRI-Ala (GRI-Ala), pcoPG4 GRII-Ala (GRII-Ala), or pcoPG4 GRIII-Ala (GRIII-Ala). As a control, cells were transfected with pMD9, pcoPE, pcoPG4, and pcoPP2 (iRT).

The qPCR results point to a problem in reverse transcription of the packaged RNA genomes in mutant virions. Because RT is present in the mutant viral particles in wild-type amounts (Fig. 1D and E), we hypothesized that the incorporated RT could be enzymatically inactive. To address particle-associated exogenous enzymatic RT activity, we used a commercially available C-type RT activity assay as reported previously (15, 26). The behavior of the iRT mutant was also examined. All RT activity values were normalized for particle-associated RT levels determined in parallel by quantitative Western blotting. Relative RT activities compared to those of the wild type were about 70 to 110% for GR box deletion mutants and about 50 to 90% for GR box alanine substitution mutations (Fig. 5B).

Taken together, GR box deletion and substitution mutants package nearly wild-type levels of genomic RNA. Furthermore, all mutants show only marginally reduced particle-associated exogenous RT activity when analyzed in vitro. In contrast, DNA content is reduced 25- to 80-fold for GR box deletion mutants and below the detection limit for GR box alanine substitutions. The infectivity of the mutants therefore correlates well with the respective DNA content but not RNA content or exogenous RT activity.

Particle morphology.

Correct reverse transcription was reported to be dependent on correct capsid assembly for HIV-1 and PFV (7, 26, 31). Therefore, RT incorporated into the PFV Gag GR box mutants examined in this study might be incapable of reverse transcribing the viral genome due to an improper microenvironment such as aberrant particle morphology.

To determine whether the observed defects in reverse transcription might be explained by defects in particle morphology, 293T cells transiently cotransfected with expression vectors for PFV Env, PFV Pol, a packageable vector RNA, and PFV Gag GR box mutants were examined by electron microscopy. Cells expressing the wild-type PFV Gag protein demonstrated budding and release of homogenous PFV particles with the typical immature capsid morphology and prominent Env spike structures on the surface (Fig. 6A and B).

FIG. 6.

Electron microscopy analysis of transfected 293T cells. Electron micrographs showing representative thin sections of transiently transfected 239T cells using a four-plasmid vector system with expression constructs for the following Gag proteins: wt (A and B), ΔGRI (C and I), ΔGRII (D), ΔGRIII (E), GRI-Ala (F and J), GRII-Ala (G), GRIII-Ala (H), or GRII-Ala (K and L). Magnifications: ×30,300 (A), ×50,000 (B, C, D, E, F, G, and H), ×37,600 (I and J), ×16,000 (K), and ×50,000 (L). Scale bar, 200 nm.

The ΔGRI mutant showed aberrant budding structures with electron-dense layers, probably representing aggregated Gag proteins, directly below the membrane at locations where the typical prominent PFV Env spike structures could be observed (Fig. 6C). In addition, particle-like structures with Env spike structures but aberrant and heterogeneously sized capsid structures were observed (Fig. 6I). The ΔGRII mutant displayed wt-like particle morphology and budding structures (Fig. 6D). ΔGRIII mutant particles also displayed wt-like morphology, with particles homogenous in size and shape. However, the lumen enclosed by the electron-dense capsid structure appeared to be slightly larger than for the wild type due to a lack of electron-dense material inside the capsid ring structure (Fig. 6E).

Analysis of the GRI-Ala mutant revealed an even more severe phenotype compared to the ΔGRI mutant. Aberrant budding structures composed of large layers of electron-dense material directly below the membrane at patches containing PFV Env spike structures were observed (Fig. 6F). Regions that appeared to be areas of budding with curved membranes and accumulated electron-dense material but no wt-like particle structures were present (Fig. 6J). The GRII-Ala mutant had budding structures with typical PFV capsid morphology and prominent Env spike structures (Fig. 6G). However, in the cytoplasm of the transfected cells large electron-dense aggregates were found, probably a paracrystalline array of viral protein (Fig. 6K). These aggregates were often found directly below the budding structures and were frequently associated with incompletely closed capsids (Fig. 6L). Such structures were not observed in any other sample examined. Cells expressing the GRIII-Ala mutant demonstrated budding and release of wt-like PFV particles that were somewhat more heterogeneous in shape or sometimes incompletely closed but showed typical Env spike structures on the surface and the typical immature capsid morphology (Fig. 6H).

Therefore, ultrastructural analysis revealed profound morphological defects for the ΔGRI as well as the GRI-Ala mutant, and slight aberrations were found for the GRII-Ala and GRIII-Ala mutants. In summary, this analysis correlates with the infectivity and genetic analysis as mutants in GRI displayed the most severe phenotype of the three GR boxes, and for each GR box the substitution mutant displayed a more severe phenotype than the deletion mutant.

DISCUSSION

In contrast to the orthoretroviruses, the PFV Gag C-terminal NC domain lacks the Cys-His motifs involved in different steps of viral replication. Instead, three GR boxes are found in the C-terminal region of PFV Gag. Most of the previous studies on GR box function were conducted in the proviral context (3, 21, 36, 38, 41, 44) in which viral nucleic acid sequence and nucleic acid secondary structure are intrinsically linked to viral protein expression, protein conformation, and protein function. Therefore, mutations result in very complex phenotypes. This complexity is best illustrated for GRI, where an internal deletion mutant led to diminished Pol encapsidation and strongly increased cellular Pol expression while a substitution mutant abolished Pol encapsidation but did not alter cellular Pol expression (21). In another study, Pol expression was strongly decreased in proviral C-terminal Gag truncations because premature stop codons led to decreased levels of spliced pol mRNA (21, 38).

In the present study we therefore characterized the role of all three GR boxes in PFV Gag in parallel using full-length deletions and substitutions employing a four-plasmid vector system. In addition, we expanded the analysis of PFV Gag GR box mutants to particle-associated DNA content and exogenous RT activity as well as viral morphology (results are summarized in Table 1).

TABLE 1.

Summary of GR box analysis^a

^aAll values are expressed relative to wt values. Differences between means of the wt and the individual mutant values were analyzed by Welch's t test. ***, P < 0.01.

^bValues are normalized for cellular expression.

^cValues are normalized to capsid protein release.

^dValues are normalized to RT protein release.

^eThe presence (+) or absence (−) of nuclear localization in transfected cells at 48h p.t.

^fExamples of ultrastructural images of mutant PFV Gag particles are shown.

This first comprehensive, systematic, and simultaneous characterization of all three Gag GR boxes in a four-plasmid vector system supports some results from earlier studies using proviral expression constructs as it indicates that the PFV Gag GR boxes have no significant influence on the physical amounts of viral particles released (44).

In the proviral context, mutations in GRI were found to abolish Pol packaging despite strong overexpression of Pol in the transfected cells of a ΔGRI mutant (21). In contrast, by using a replication-deficient four-plasmid expression-optimized vector system, Pol was packaged into viral particles of all GR box mutants. Mature Pol subunits were found to be resistant to subtilisin digestion and therefore are most likely particle associated. Furthermore, their enzymatic functions were intact, as indicated by Gag and Pol processing as well as only marginally reduced particle-associated in vitro RT activity. At this time we can only conclude that intact GR boxes are nonessential for Pol packaging in the four-plasmid vector system. Nevertheless, the situation might be different in the proviral system, possibly due to superimposition of independent effects or involvement of currently unknown factors.

In line with previous studies the infectious titers were severely reduced for all deletion mutants, indicating a specific infectivity defect in mutant virions. Surprisingly, alanine substitution mutants showed no detectable infectivity. This seemed at first somewhat counterintuitive as deletion of a motif often leads to a more severe phenotype than does its substitution, probably because of a reduced effect on protein folding. Interestingly, similar effects have been found in previous studies of PFV Gag in the proviral context, where replacing GRII by an HA tag proved to be noninfectious while a revertant with a large deletion in this region regained infectivity (44). In addition, a detailed study of GRI using proviral constructs found 0.1% of wild-type infectivity for a mutant carrying an 11-aa deletion while replacing these 11 amino acids with an HA tag or a short alanine stretch led to no detectable infectivity (21). Although we cannot explain this discrepancy between deletion and heterologous substitution mutants, it seems to be systematic.

We demonstrate that the reason for the infectivity defect of substitution mutants is the absence of viral DNA in the particles, apparently caused by the failure to reverse transcribe the packaged viral RNA.

However, the length and specific sequence used for substitutions also directly contribute to the severity of this phenomenon. The phenotypes of two alanine substitution mutants of GRI and GRII harboring only four alanines each, which we obtained inadvertently, support this conclusion. These displayed an intermediate phenotype with 0.7% (GRI-Ala4) and 3.3% (GRII-Ala4) of wild-type infectivity (data not shown). Furthermore, complete abolishment of viral infectivity seemed to occur only if the GR boxes were completely replaced with unrelated sequences. By replacing each GR box with another, we demonstrated that GRI and GRIII can complement each other more efficiently than GRII and vice versa. Nevertheless, even the GR box replacement mutants that seem to be less compatible (e.g., GRI-II, GRII-I, and GRII-III) still support formation of particles with higher specific infectivity than the corresponding GR box deletions or alanine substitutions. In agreement with these results, Lee et al. (21) also found that replacing GRI with sequences from GRII, GRIII, or other similar sequences in the proviral context is compatible with viral replication. However, our data on the GR box replacement mutants suggest that GRI and GRIII are more closely related than GRII because they can better functionally complement each other. This resemblance might result from sequence or functional similarities, or it might be due to a specific function of GRII, e.g., nuclear localization that cannot be replaced by GRI or GRIII.

Our results support the current notion of GRII being responsible for PFV Gag nuclear localization because only GRII mutants displayed an altered intracellular distribution. Interestingly, Gag subcellular localization was found to be independent of the expression of the other viral structural proteins and viral RNA. The function of Gag nuclear translocation, however, remains a fundamental question, particularly because GRII is the most highly conserved GR box between all FV species, but nuclear localization is observed only for primate FV Gag. It has been speculated that the CBS in GRII has a role in tethering the viral genome to the host cell chromatin in order to facilitate integration (41). However, our data do not support this notion because the decrease or total lack of detectable infectivity of the individual mutants including those affecting GRII correlated very well with the viral cDNA amounts measured in the particles and not with the intracellular distribution of the mutant protein. Therefore, it is tempting to speculate that the cDNA amounts measured can fully account for the infectious titers of the mutants. This suggests that the mutations cause no additional defects in the early stages of PFV infection.

Although an early RT step has been described previously (5, 46), FVs reverse transcribe their RNA genome to a large extent during, or shortly after, capsid assembly in the virus-producing cell (25). Released wild-type particles therefore contain either RNA or DNA. RT-qPCR analysis revealed slightly decreased levels of genomic RNA packaging for all GR box mutants examined. This is reminiscent of orthoretroviral Cys-His motif mutants (reviewed in reference 8). Though the decrease in RNA packaging was not statistically significant, it seemed that all PFV Gag GR boxes contributed to RNA encapsidation but that none of the individual GR boxes was absolutely essential.

In comparison to encapsidated genomic RNA levels, viral cDNA levels were reduced 10- to 40-fold for GR box deletion mutants, and viral cDNA was undetectable for the substitution mutants. This suggests an additional defect in reverse transcription. Because in vitro activity of reverse transcriptase was found to be only slightly decreased, in vivo activity is probably impaired by the lack of a proper microenvironment.

For orthoretroviruses, correct capsid assembly is crucial for subsequent reverse transcription of the packaged viral RNA genome. Even a mild defect in orthoretroviral capsid assembly can already adversely affect reverse transcription upon target cell entry. HIV-1 Gag mutants that display aberrant capsid morphology are associated with defects in early steps of the viral replication cycle that can lead to incomplete reverse transcription, failure to initiate reverse transcription, or premature reverse transcription (6, 7, 16, 31). For PFV it was also suggested that proper PFV core assembly is required for correct reverse transcription of the viral genome (26). For the GR box mutants examined in this study, a clear correlation between altered capsid morphology and impairment of genome reverse transcription of the individual mutants was observed only to a limited extent. The GRI deletion mutant displayed aberrant capsid structures and large electron-dense accumulations below cellular membranes. The deletion mutants of GRII and GRIII, however, displayed no obvious gross morphological defects. This is in line with ΔGRI having the lowest infectious titers of all three deletions.

Electron microscopy analysis of the of GRI mutants revealed severe morphological defects (large electron-dense accumulations at the membrane with visible Env spike structures), also suggesting reduced viral particle budding. However, biochemical data indicated wt-like particle release. To address this discrepancy, we performed additional density gradient centrifugation and rate-zonal centrifugation of ΔGRI particle preparations and compared them to wt preparations. The viral particles appeared at the same buoyant densities, and the rates of sedimentation were similar (data not shown). Furthermore, electron micrographs of target cells loaded with concentrated supernatants of ΔGRI viral particles revealed a more heterogeneous particle size and aberrant capsid morphology than observed in the corresponding wild-type samples. However, no large particulate structures with electron-dense accumulations below the membranes could be observed (data not shown). Therefore, the ΔGRI particles released were of similar quantity, size, shape and density as wt particles.

All alanine substitution mutants exhibited more serious defects in infectivity and genome reverse transcription than mutants with deletions of the respective GR box. To a certain extent this was also true for capsid morphogenesis. For GRI-Ala, no remaining wt-like budding structures were observed, GRII-Ala showed additional paracrystalline accumulations in the cytoplasm, probably of viral protein origin, and the GRIII-Ala mutant displayed somewhat more heterogeneous budding structures and particle morphology.

However, the total lack of infectivity of the alanine substitution mutants leaves some open questions. PFV Gag GRI-Ala and GRIII-Ala displayed wt-like intracellular localization. All GR box mutants yielded wt-like numbers of viral particles with wt-like protein composition and processing. In particular, Pol is packaged and enzymatically functional in vitro. Furthermore, released mutant particles contain genomic viral RNA, albeit at slightly reduced amounts, and especially for GRII-Ala and GRIII-Ala, capsid morphology is only marginally altered. It therefore seems that none of these findings can fully explain the complete lack of particle-associated viral cDNA observed. Although it is possible that the observed phenotype is a cumulative one, it seems more likely that it is caused by a so far unknown effect not addressed or not detected in this study.

The mutants produced here should provide the basis to address these newly raised questions. They have already proved to be excellent tools for the rapid and easy cloning of new constructs. Because these basic modules are also available in a C-terminal eYFP fusion context, they can expand PFV Gag GR box research to include modern sophisticated techniques such as live-cell imaging. Finally, they might easily be transferred to the proviral system to help answer some remaining questions.

Taken together, results of our study provide valuable support for the known function of PFV Gag GR boxes. However, they also question some previously described functions such as GRI-mediated Pol packaging or reduced infectivity as a result of absent chromatin tethering by GRII CBS deletion mutants (21, 41). Most importantly, the present study highlights entirely novel functions for PFV Gag GR boxes in reverse transcription and particle morphogenesis. Because this is clearly reminiscent of the Cys-His motifs in the HIV Gag NC domain, it emphasizes functional equivalency of PFV Gag GR boxes and orthoretroviral Cys-His motifs.