Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus

Abstract

The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C_377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding.IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.

Keywords: Alix; feline immunodeficiency virus; late domain; nucleocapsid.

FIG 1

Schematic representation of the HIV-1 and FIV Gag regions. Shown are the matrix (MA), capsid (CA), and nucleocapsid (NC) domains, as well as the HIV-1 p2, p1, and p6 peptides and the FIV p1 and p2 domains. The amino acid sequences of the HIV-1 and FIV NC domains are also shown. The HIV-1 and FIV regions containing the recognized viral L-domains (PTAP and YPLASL for HIV-1; PSAP for FIV) are underlined, as is the FIV LLDL motif. The HIV-1 NY5/BRU (LAV-1) recombinant clone pNL4-3 (GenBank accession no. M19921) and the FIV Petaluma strain, clone 34TF10 (GenBank accession no. M25381), were used as references.

FIG 2

Alix functions in FIV budding independently of the entire Gag p2 domain and independently of the presence of a binding motif. (A and B) 293T cells were transfected with plasmids expressing wild-type FIV Gag/Pol (PSAPx₅LLDL) or an FIV Gag/Pol variant either with a mutation in the PSAP motif (ASAAx₅LLDL), lacking the entire Gag p2 region (Δp2), or combining the PSAP inactivation with a YPDL motif replacing the conserved LLDL domain (ASAAx₅YPDL). (A) Some of the samples (+) were also cotransfected with a construct expressing an HA-tagged version of Alix. Twenty-four hours posttransfection, VLPs were spun down by ultracentrifugation and were subjected to Western blot analysis by employing an anti-FIV capsid Gag antibody (p24). Cell lysates (Cells) were also assayed with α-p24 or with an antibody specific for the HA tag (α-HA). C– stands for mock-transfected cells. Asterisks point to capsid-containing intermediate products of the Gag polyprotein (Pr50) cleavage process (likely MA-CA-p1-NC, MA-CA-p1/CA-p1-NC-p2). The arrowhead in the lane for mock-transfected cells indicates a cross-reactive protein that is also visible in the Δp2 lanes. Arrows point to partially processed Gag intermediates present in the cell supernatants of the rescued Δp2 and ASAAx₅YPDL mutants. Numbers indicate molecular size markers (in thousands). (B) Graph showing the relative virus release efficiency, calculated as the ratio of virion-associated Gag to total Gag (cells plus VLPs), normalized to FIV Gag/Pol PSAPx₅LLDL release (26). Values are averages calculated from at least three independent experiments, and error bars indicate standard deviations. Band pixel intensity was quantified by using the software provided with the Alliance Q9 advanced chemiluminescence imager (Uvitec; Cleaver Scientific).

FIG 3

Overexpression of the Alix Bro1 domain is sufficient to rescue FIV Δp2 budding. (A) Schematic representation of the Alix mutants employed in this study. (B) 293T cells were transfected with the construct encoding FIV Δp2 (+) along with a plasmid expressing either wild-type Alix (WT) or the indicated mutant (Bro1 or VPRD), each fused to an HA tag. Twenty-four hours posttransfection, VLPs were spun down by ultracentrifugation and were analyzed by Western blotting with the α-p24 antibody (VLPs). Cell lysates (Cells) were also assayed with α-p24 (right) and α-HA (left) antibodies. C– stands for cells transfected with the corresponding empty vector (in place of an Alix-expressing plasmid) along with the construct encoding FIV Δp2. The results shown have been replicated in at least three independent experiments. Numbers indicate molecular weight markers (in thousands). (C) Graph showing the relative virus release efficiency, calculated as the ratio of virion-associated Gag to total Gag (cells plus VLPs), normalized to the release of FIV Δp2 plus WT Alix. Values are averages calculated from at least three independent experiments, and error bars indicate standard deviations. Band pixel intensity was quantified by using the software provided with the Alliance Q9 advanced chemiluminescence imager (Uvitec; Cleaver Scientific).

FIG 4

C₃₉₀ and C₄₀₉ are not involved in the ability of Alix to rescue the budding of PSAP-deficient FIV. 293T cells were transfected with the PSAPx₅LLDL or C_390,409S/PSAP construct (A) or with the ASAAx₅LLDL or C_390,409S/ASAA plasmid (B). In the experiment for which results are shown in panel B, cells were also transfected with a construct expressing HA-Alix (+) or with the corresponding empty vector (–). Twenty-four hours posttransfection, VLPs were spun down by ultracentrifugation and were analyzed by Western blotting by employing the α-p24 antibody. Cell lysates (Cells) were also assayed with α-p24 (A and B) and α-HA (B) antibodies. The results shown have been replicated in at least three independent experiments. Numbers indicate molecular weight markers (in thousands). (C) Graphs show the relative virus release efficiency, calculated as the ratio of virion-associated Gag to total Gag (cells plus VLPs), normalized to the release of FIV PSAPx₅LLDL (left) or FIV ASAAx₅LLDL (right). Values are averages calculated from at least three independent experiments, and error bars indicate standard deviations. Band pixel intensity was quantified by using the software provided with the Alliance Q9 advanced chemiluminescence imager (Uvitec; Cleaver Scientific).

FIG 5

Neither C₃₉₀ and C₄₀₉ nor the PSAP motif nor the LLDL domain is the main determinant in the interaction of Alix with FIV Gag. 293T cells were transfected with constructs expressing either PSAPx₅LLDL/STOP, C_390,409S/ASAA/STOP, or C_390,409S/PSAPx₅AAAA/STOP along with a plasmid encoding HA-Alix (+) or the corresponding empty vector (–). Forty-eight hours posttransfection, similar amounts of VLPs were analyzed by Western blotting, using either the α-HA (upper panels) or the α-p24 (lower panels) antibody. Cell lysates (Cells) were also assayed.