Molecular characterization of feline immunodeficiency virus budding

Abstract

Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5') each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.

FIG. 1.

A highly conserved P(T/S)AP motif in FIV Gag is required for FIV release and replication. (A) An alignment of C-terminal sequences from lentiviral Gag proteins is shown, with the highly conserved P(T/S)APP motif highlighted (GenBank; National Center for Biotechnology Information; Entrez Genome). Abbreviations: SIVagm, SIV from African green monkey; Visna, visna/maedi lentivirus; BIV, bovine immunodeficiency virus. The host species of each virus is indicated. Sites of protease cleavage in the C-terminal domain of Gag liberating HIV-1 p6 and FIV p2 are marked with an arrow, resulting in the proposed numbering of amino acids in FIV p2 (17, 34, 65). Domains of FIV Gag and residues altered by site-directed mutagenesis in the present study are shown. (B) The PSAP motif in FIV Gag (p2) is required for efficient virus release. HeLa cells were transfected with an FIV-expression vector (FP93) and metabolically radiolabeled with [³⁵S]Met/Cys. FIV proteins were detected in cell or virus fractions by immunoprecipitation with anti-FIVp24gag and resolved by SDS-PAGE. p50, full-length Gag precursor protein (MA-CA-p1-NC-p2); Gag processing intermediates p47, p40, and p33; p24, capsid (CA). The ∼45-kDa band detected in mock-transfected cells is a cross-reactive protein. Gag protein levels were quantified by phosphorimager analysis, and the relative virus release efficiency was calculated as the ratio of virion-associated Gag to total Gag (cells plus virus), normalized to the WT FIV release, and the results were averaged from at least three independent experiments. Error bars indicate the standard deviations (SD). (C) CrFK cells were infected with RT-normalized WT or mutant FIV(Orf2rep) pseudotyped with VSV-G. The RT levels in culture supernatants were measured prior to each cell passage.

FIG. 2.

Effect of PSAP⁻ and LLDL⁻ mutations on the morphogenesis of FIV budding. HeLa (left panels) or CrFK (right panels) cells were transfected with plasmids expressing WT or mutant FIV Gag and then fixed 2 days posttransfection. Typical images of both normal and defective virions and budding intermediates found associated with the plasma membrane are shown. All scale bars represent 100 nm unless indicated otherwise.

FIG. 3.

FIV release is sensitive to dominant-negative disruption of Tsg101 and Vps4A. (A) HeLa cells were mock transfected (lane “−”) or were cotransfected with FIV expression vector (FP93) and pBS empty vector (lane “+”), or expression vectors encoding TSG-F (lane F), TSG-5′ (lane 5′), TSG-3′ (lane 3′) or Vps4A(E228Q)-eGFP (lane V). Transfected cells were metabolically radiolabeled with [³⁵S]Met/Cys; FIV proteins were detected in cell or virus fractions by immunoprecipitation with anti-FIVp24gag and resolved by SDS-PAGE. FIV Gag products p50, p40, p33, and p24 are described in the Fig. 1 legend. Relative virus release was determined as indicated in the Fig. 1 legend based on data obtained from five independent experiments ± SD. FIV release was found to be significantly different from the negative control (pBS) in all experimental samples by a two-tailed one-sample t test (P < 0.05). (B) HeLa or CrFK cells were transfected with the same vectors used in virus release assays described for panel A. Exogenous Tsg101 was visualized by immunofluorescence using a rabbit anti-HA antibody (red); Vps4EQ was visualized directly via its green fluorescent protein tag (green).

FIG. 4.

Full-length and truncated forms of Tsg101 disrupt FIV budding. HeLa and CrFK cells were transfected with FIV expression vector alone (FIV only) or were cotransfected with expression vectors encoding TSG-F, TSG-5′, or TSG-3′. Cells were fixed for EM 1 day posttransfection. Typical images of both normal and defective virions and budding intermediates associated with the plasma membrane are shown. Scale bars represent 100 nm, unless indicated otherwise.

FIG. 5.

FIV release and replication are inhibited in CrFK cells by stable TSG-5′ expression. (A, inset) Cell lysates from control CrFK(zeo) or TSG-5′-expressing cells [CrFK/TSG-5′(zeo)] at 4 months posttransfection were subjected to SDS-PAGE and immunoblotted with rabbit anti-HA antibody to detect stably expressed TSG-5′ protein. (A) Control [CrFK(zeo)] or TSG-5′(zeo)-expressing cells were infected with 10-fold serial dilutions (10⁵ to 10⁷ RT cpm) of cell-free WT FIV (34TF10). RT activity in cultured supernatants was determined prior to each cell passage. (B) Control CrFK(zeo) or TSG-5′(zeo)-expressing cells were transfected with pFIV-34TF10 and fixed for EM at 2 days posttransfection. Typical lentiviral particles associated with the plasma membrane are shown. Scale bars, 100 nm. (C) FIV release assays in control [CrFK(zeo)] or TSG-5′(zeo)-expressing cells. The results were averaged from five independent experiments.

FIG. 6.

Tsg101 depletion inhibits FIV but not EIAV release. (A) HeLa cells were serially transfected with siRNA at both 0 and 24 h and with the TSG-F expression vector at 24 h. Cell lysates, prepared at 48 h, were subjected to SDS-PAGE and immunoblotted with anti-HA antiserum. Exogenous Tsg101 expression was undetectable after cotransfection with as little as 2.5 nM Tsg101-specific siRNA but not with negative control siRNAs. In contrast, levels of endogenous Alix were not affected by Tsg101 siRNA. (B) HeLa cells were transfected with FIV or EIAV expression vectors in the absence of siRNA (−) or in the presence of 5 nM control siRNA (neg.) or Tsg101-specific siRNA. Transfected cells were metabolically radiolabeled with [³⁵S]Met/Cys. FIV and EIAV proteins were detected in cell and virus fractions by immunoprecipitation with anti-FIVp24gag (FIV) or anti-EIAV horse antiserum (EIAV) and resolved by SDS-PAGE. FIV Gag products p50, p40, p33, and p24 are described in the Fig. 1 legend. In EIAV immunoprecipitations, p55 is the 55-kDa EIAV Gag precursor. Relative virus release efficiency was determined as indicated in the Fig. 1 legend, based on data obtained from three independent experiments, ± the SD.

FIG. 7.

Dominant-negative Alix V domain fragment inhibits the release of HIV-1 and EIAV, but not that of FIV. The domain structure of Alix protein is shown (top), with the Bro1, “V” (amino acids 364 to 716), and C-terminal proline-rich domains indicated. HeLa cells were cotransfected with expression vectors for EIAV, HIV-1, or FIV and empty vector (lanes “−”) or plasmid expressing the Alix V domain (lanes “+”). Transfected cells were metabolically radiolabeled with [³⁵S]Met/Cys. EIAV, HIV-1, and FIV proteins were detected in cell and virus fractions by immunoprecipitation with anti-EIAV horse antiserum (EIAV), HIV-Ig (HIV-1), or anti-FIVp24gag (FIV) and were resolved by SDS-PAGE. FIV Gag products p50, p40, p33, and p24 are described in the Fig. 1 legend. In EIAV samples, p55 denotes the 55-kDa EIAV Gag precursor. In HIV-1 samples, the Gag precursor protein Pr55^Gag (p55) and the mature CA protein (p24) are indicated. Averages of relative virus release efficiency, determined as indicated in the Fig. 1 legend, were based on data obtained from three independent experiments (for FIV and HIV-1) ± the SD and one representative experiment for EIAV.

FIG. 8.

Analysis of Alix-V and Tsg101-UEV binding to peptides derived from FIV, HIV-1, and EIAV Gag by fluorescence anisotropy. (A) Alix-V protein binds to peptides derived from HIV-1 and EIAV Gag, but not FIV Gag. (B) Tsg101-UEV protein binds to FIV and HIV-1 Gag, but not EIAV Gag. Protein-peptide interactions were detected by fluorescence anisotropy upon the addition of increasing amounts of either purified Alix-V protein (A) or purified Tsg101-UEV protein (B) to a fixed concentration (50 nM) of FITC-labeled peptides, based on FIV, EIAV, or HIV-1 Gag C-terminal sequences. Calculated dissociation constants (K_d) are indicated.