Prototype foamy virus protease activity is essential for intraparticle reverse transcription initiation but not absolutely required for uncoating upon host cell entry

Abstract

Foamy 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.

Fig 1

PFV production systems. Schematic illustration of the different PFV production systems used in the study. Three systems based on authentic PFV ORFs were utilized and include systems for production of the following: prov, replication-competent virus based on CMV (cytomegalovirus)-driven proviral expression constructs (A); 3-nco, replication-deficient vectors based on CMV-driven Gag/Pol and Env packaging constructs and CMV-driven PFV transfer vector (PTV) containing an egfp marker gene expression cassette (B); and 4-nco, replication-deficient vectors based on separate CMV-driven packaging vectors for Gag, Pol, and Env and the previously mentioned transfer vector (C). (D) A fourth replication-deficient vector system (4-co) is based on separate packaging constructs with expression-optimized ORFs encoding PFV Gag, Pol, and Env and the same transfer vector mentioned above. R, long terminal repeat region (LTR); U5, LTR unique 5′ region; U3, LTR unique 3′ region; ΔU3: enhancer-promoter deleted U3 region; IP, internal promoter; CAS, cis-acting sequence; SFFV, spleen focus-forming virus U3 promoter; SD, splice donor; SA, splice acceptor; PR, protease domain; RT, reverse transcriptase domain; L, linker domain between PR and RT; IN: integrase domain; pA: bovine growth hormone polyadenylation site. Major PFV PR cleavage sites in PFV Gag and Pol are indicated by black arrows; iPR (D₂₄A), iRT (DD_312–315GAAA), and iIN (D₉₃₆A) mutations are indicated with stars throughout the pol ORF; authentic ORFs are indicated by boxes with continuous lines; expression-optimized ORFs are indicated by boxes with dashed lines. Numbers indicate amino acid positions in Pol.

Fig 2

Comparison of the features of the different PFV production systems. 293T cells were transiently transfected with the components of the different PFV production systems as indicated. The proviral constructs (prov) were cotransfected with the EGFP-expressing PFV transfer vector puc2MD9 at a ratio of 1:1. (A) Cell-free viral supernatants of the wild-type controls of the individual PFV production systems were titrated on HT1080 target cells using a flow cytometric marker gene assay, and titers were determined at 24 to 72 postransduction. For the proviral constructs, similar titers were obtained when they were not cotransfected with puc2MD9 and titrated on HT1080 PLNE cells, containing a Tas-inducible nuclear EGFP expression cassette under the control of the PFV LTR (data not shown). ffu, focus-forming units. (B to E) Cell lysates (cell) as well as viral particle preparations (virus), concentrated by ultracentrifugation through 20% sucrose, were analyzed by Western blotting. Serial dilutions of the samples of the expression-optimized four-component vector system (4-co, lanes 5 to 9 and 14 to 18) were loaded to determine their relative expression levels in comparison to the other PFV production systems. undil, undiluted. (B) Rabbit polyclonal antibodies specific for PFV Gag (α-Gag). (C) Mouse monoclonal antibody mixture specific for PFV PR and PFV IN (α-PR+α-IN). (D) Rabbit polyclonal antibodies specific for PFV Env-LP (α-Env-LP). (E) Mouse monoclonal antibodies specific for GAPDH (α-GAPDH).

Fig 3

Infectious PFV vector particles generated by separate PFV Pol subunit packaging constructs. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9 (for lanes 4, 13, and 14, the amount was reduced to 2/3), identical amounts of Gag and Env packaging vectors pcoPG4 and pcoPE, and different amounts (starting with 0.91 μg of DNA per 10-cm dish as relative amount 1; lanes 1, 11, and 12) of the various Pol packaging constructs. Pol packaging constructs used contained expression-optimized ORFs for the full-length wild-type PFV Pol protein (coPP; lanes 1, 11, and 12) or variants thereof with enzymatically inactive reverse transcriptase (coPP iRT; lane 7) or integrase (coPP iIN; lane 8), as indicated. Alternatively, packaging constructs harboring expression-optimized ORFs for the mature PR-RT (coRT) or IN (coIN) subunit were cotransfected together (coRT+coIN, lanes 2 to 4, 13, and 14) or alone (coRT, lanes 5, 17, and 18; coIN, lanes 6, 15, and 16), as indicated. As a control, the Env packaging construct was omitted in a sample containing the wild-type full-length Pol packaging construct (coPP+ΔEnv, lanes 9, 19, and 20), or cells were transfected only with pUC19 DNA (mock, lanes 10, 21, and 22). The relative amount (rel. amount) of the individual Pol packaging construct used in comparison to the full-length wild-type PFV Pol protein (coPP)-encoding construct is indicated on top. The total amount of transfected DNA (16 μg) was kept constant in all samples by addition of pUC19 DNA. (A to E) Proteins of viral particle samples (virus) purified by ultracentrifugation through 20% sucrose and cell lysates (cell) harvested 48 h posttransfection. For the experiments shown in panels C to E, samples were either digested with subtilisin (+) or mock incubated (−) prior to lysis. Subsequently, samples were separated by SDS-PAGE and analyzed by Western blotting using mouse monoclonal antibody mixture specific for PFV PR and PFV IN (α-PR+α-IN) (A and D), rabbit polyclonal antibodies specific for PFV Gag (α-Gag) (B and E), or rabbit polyclonal antibodies specific for PFV Env-LP (α-Env-LP) or mouse monoclonal antibodies specific for PFV Env-SU (α-Env-SU) (C). The identity of the individual proteins is indicated on the right. (F) Three days postinfection, relative infectivities of 293T cell culture supernatants were determined with an egfp marker gene transfer assay. The values obtained using full-length expression-optimized pol ORF expression plasmid (coPP, column 1) were arbitrarily set to 100%. Sample values were normalized for capsid protein release. Absolute titers of these plain supernatants were (4.31 ± 0.75) × 10⁶ EGFP-positive focus-forming units (FFU)/ml. Means and standard deviations of four independent experiments are shown. (G) 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 DNA copies was determined in comparison to the wild type by qPCR. The mean values and standard deviations of the relative RNA and DNA contents of at least three independent experiments are shown. Sample values were normalized for capsid protein release. Differences between means of the wild type and the individual mutants were analyzed by Welch's t test (*, P < 0.01).

Fig 4

Biochemical analysis of PFV particles derived from Pol packaging constructs with enzymatically inactive PR domains. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9, Env packaging vector pcoPE, different ratios of expression-optimized PFV Gag packaging constructs coding for p71^Gag and p68^Gag as indicated, and various Pol packaging constructs harboring expression-optimized ORFs for the following: the full-length wild-type PFV Pol protein (coPP, lanes 1, 2, 12 to 15, 25, and 26); a variant thereof with enzymatically inactive PR (coPP iPR, lanes 3, 4, and 16 to 19) or enzymatically inactive RT (coPP iRT, lanes 9, 31, and 32), the mature PR-RT and IN subunits (coRT+coIN, lanes 5, 6, and 20 to 23); the mature PR-RT subunit with enzymatically inactive PR and IN subunit (coRT iPR+coIN, lanes 7, 8, and 27 to 30). As a control, the Env packaging construct was omitted in a sample containing the wild-type full-length Pol packaging construct (coPP+ΔEnv, lanes 10, 33, and 34), or cells were transfected only with pUC19 DNA (mock, lanes 11 and 24). The total amount of transfected PFV Gag packaging construct was kept constant in all samples. The ratios of p71^Gag (p71) to p68^Gag (p68) packaging constructs are indicated on top. Western blot analysis of cell lysates (cell) and pelleted viral supernatants (virus) digested with subtilisin (+) or mock (−) incubated using a mouse monoclonal antibody mixture specific for PFV PR and PFV IN (α-PR+α-IN) (A), rabbit polyclonal antibodies specific for PFV Gag (α-Gag) (B), rabbit polyclonal antibodies specific for PFV Env-LP (α-Env-LP) (C), or mouse monoclonal antibodies specific for PFV Env-SU (α-Env-SU) (D). The identities of the individual proteins are indicated on the right.

Fig 5

Infectious PFV particles derived from Pol packaging constructs with enzymatically inactive PR domains. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9, Env packaging vector pcoPE, and different ratios of expression-optimized PFV Gag packaging constructs coding for p71^Gag and p68^Gag (indicated below the x axis) as well as various Pol packaging constructs harboring expression-optimized ORFs for the full-length wild-type PFV Pol protein (coPP), a variant thereof with enzymatically inactive PR (coPPiPR), the mature PR-RT and IN subunits (coRT+coIN), or the mature PR-RT subunit with enzymatically inactive PR and IN subunit (coRTiPR+coIN) as indicated. At 3 days postinfection, relative infectivities of extracellular 293T cell culture supernatants were determined with an egfp marker gene transfer assay. The values obtained using a full-length expression-optimized pol ORF expression plasmid (coPP) were arbitrarily set to 100%. Absolute titers of these plain supernatants were (4.99 ± 2.74) × 10⁶ EGFP-positive FFU/ml. Means and standard deviations of at least four independent experiments are shown.

Fig 6

Dependence of viral infectivity on PR structure. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9, Env packaging vector pcoPE, and various Pol packaging constructs harboring expression-optimized ORFs for the full-length wild-type PFV Pol protein (coPP, lanes 1, 10, and 11), the mature PR-RT and IN subunits (coRT+coIN, lanes 2, 12, and 13), the mature PR-RT subunit with enzymatically inactive PR and IN subunit (coRT iPR+coIN, lanes 3, 14, and 15), the full PR domain-deleted PR-RT subunit and IN subunit (coRT1+coIN, lanes 4, 5, and 16 to 19), and the core PR-deleted PR-RT subunit and IN subunit (coRT2+coIN, lanes 6, 7, and 20 to 23) as well as different ratios of expression-optimized PFV Gag packaging constructs coding for p71^Gag and p68^Gag as indicated. As a control, the Env packaging construct was omitted in a sample containing the wild-type full-length Pol packaging construct (coPP +ΔEnv, lanes 9, 26, and 27), or cells were transfected only with pUC19 DNA (mock, lanes 8, 24, and 25). The total amount of transfected PFV Gag packaging was kept constant in all samples. The ratios of p71^Gag (p71) to p68^Gag (p68) packaging constructs are indicated on top. The total amount of transfected DNA (16 μg) was kept constant in all samples by addition of pUC19 DNA. (A to C) Western blot analysis of Pol and Gag expression in cell lysates (cell) and concentrated virus (virus) either digested with subtilisin (+) or mock (−) incubated using a polyclonal antibody mixture specific for SFV-1 PR-RT and PFV IN (α-RT+α-IN) (A), a rabbit polyclonal antibody specific for PFV Gag (α-Gag) (B), and a rabbit polyclonal antibody specific for PFV Env-LP (α-Env-LP) or mouse monoclonal antibodies specific for PFV Env-SU (α-Env-SU) (C). (D) At 3 days postinfection, transduction rates of HT1080 cells were measured using an egfp marker gene transfer assay. The values obtained using full-length expression-optimized pol ORF expression plasmid (coPP) were arbitrarily set to 100%. Absolute titers of these plain supernatants were (4.38 ± 0.96) × 10⁶ EGFP-positive FFU/ml. Means and standard deviations of at least four independent experiments are shown. In this assay the sample coPP+ΔEnv was not determined (nd).

Fig 7

Correlation of released particle-associated nucleic acid composition with Gag processing and Pol enzymatic activities. 293T cells were cotransfected with identical amounts of PFV transfer vector puc2MD9, Env packaging vector pcoPE, and various Pol packaging constructs harboring expression-optimized ORFs for the full-length wild-type PFV Pol protein (coPP), a variant thereof with enzymatically inactive PR (coPP iPR), or enzymatically inactive RT (coPP iRT), the mature PR-RT and IN subunits (coRT+coIN), the mature PR-RT subunit with enzymatically inactive PR and IN subunit (coRT iPR+coIN), and different ratios of expression-optimized PFV Gag packaging constructs coding for p71^Gag or p68^Gag as indicated. As a control the Env packaging construct was omitted in a sample containing the wild-type full-length Pol packaging construct (coPP+ΔEnv), or cells were transfected only with pUC19 DNA (mock). The total amount of transfected PFV Gag packaging was kept constant in all samples. The ratios of p71^Gag (p71) to p68^Gag (p68) packaging constructs are indicated below the x axis. The relative nucleic acid composition of mutant particles was 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 DNA copies was determined in comparison to the wild type by qPCR. The mean values and standard deviations of the relative RNA and DNA contents of at least three independent experiments are shown. Sample values were normalized for capsid protein release. Differences between means of the wild type and the individual mutants were analyzed by Welch's t test (* P < 0.01).

Fig 8

PR-deficient, infectious PFV particles generated by production systems with authentic-codon usage. 293T cells were transiently transfected with the components of the different PFV production systems as indicated. The proviral constructs (prov) were cotransfected with the EGFP-expressing PFV transfer vector puc2MD9 (PTV) at a ratio of 1:1. Cell-free viral supernatants of various combinations of Gag and Pol mutants in the context of the individual PFV production systems were titrated on HT1080 target cells using a flow cytometric marker gene assay, and titers were determined at 72 posttransduction. For the proviral constructs, similar titers were obtained when they were not cotransfected with puc2MD9 and titrated on HT1080 PLNE cells, containing a Tas-inducible nuclear EGFP expression cassette under the control of the PFV LTR (data not shown). The values obtained using full-length gag (p71) and pol ORFs with an enzymatically active PR domain (Pol) were arbitrarily set to 100%. Absolute titers of these plain supernatants are shown in Fig. 2A. Means and standard deviations of at least three independent experiments are shown.