CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

Abstract

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.

Fig 1. Localization of CD2v around the viral factory during ASFV infection.

A: ASFV-E70 infected COS cells were immune stained with α-P72 to detect the viral factory, and with α-CD2v after 16 hpi. B: COS cells were previously transfected with HA-CD2v for 24 hpt, before being infected with the ASFV-NHV strain, which does not express CD2v, for 16 hpi, and immune stained with α-p72 and with α-CD2v. C: ASFV-E70 infected COS cells were immune stained with α-vimentin and α-CD2v after 16 hpi. Images of apical and medial slides are shown in the upper and lower panels respectively.

Fig 2. CD2v colocalized with AP-1 in both infected and transfected cells.

A: COS cells were MOCK-infected or infected with ASFV-E70 and immune-stained 16 hpi with α-γ-adaptin (AP-1) and α-CD2v. B: COS cells were MOCK-infected or infected with ASFV-E70 for 6h, and then treated with BFA for 3 h. At 9 hpi, cells were immune-stained as in A.

Fig 3. Both full length CD2v and the Ct domain colocalize with AP-1 in COS transfected cells.

A: schematic constructs of CD2v-GFP. COS cells were transfected either with the full length CD2v (B), the CD2v Ct domain (C) of GFP fusion proteins or the GFP vector (D). 24 hpt cells were stained with α-γ-adaptin (AP-1) and AP-1 and GFP fusion proteins were observed.

Fig 4. CD2v interacting with AP-1 during ASFV infection.

COS cells were MOCK-infected or infected with ASFV-E70 in the presence or not of tunicamycin (5 μg/ml). After 16 hpi, cells were lysed and immune precipitated with α-γ-adaptin (AP-1) as explained in Material and Methods. In ASFV infected cells (left panel, Input) bands corresponding to the glycosylated (110kDa) and non-glycosylated CD2v (42kDa) full length forms of CD2v and the predicted cleaved CD2v Ct domain (26kDa) were observed. In the right panel (IP) the immunoprecipitated and coimmunoprecipitated AP-1 and CD2v are indicated.

Fig 5. CD2v di-Leu motif is functionally interchangeable with the HIV-Nef di-Leu motif in the AP-1 binding assay.

COS cells were lysed and incubated with immobilized GST-Nef-WT, GST-Nef-LLAA, GST-Nef-diLeuCD2v or GST alone and coprecipitated with glutathione-Sepharose beads, separated by 10% SDS-PAGE followed by immunoblotting with an anti-γ-adaptin (AP-1) antibody. Densitometry values of anti-AP-1 bands relative to anti-GST are presented in the graph below. A representative experiment of at least three independent experiments is shown.

Fig 6. The di-Leu motif is not involved in CD2v-AP-1 co-localization.

(A): COS cells were transfected with either wild-type CD2v-GFP or LLAA mutant. 24 hpt, the localization of both CD2v and AP-1 (immune stained with α-γ-adaptin) was examined by confocal microscopy. (B): COS cells were transfected with either wild-type HA-CD2v or LLAA mutant and then infected for 16 h with the ASFV-NHV. CD2v and AP-1 were immune stained with α-CD2v and α-γ-adaptin respectively and examined by confocal microscopy.

Fig 7. The CD2v-AP-1 interaction is mediated by the Ct domain of CD2v but not by the CD2v di-Leu motif.

(A): Structure of GST-fusion proteins of different Ct regions of CD2v: Ct domain of CD2v (240–402 aa), wild-type (CD2v WT), or the di-Leu mutant (CD2v LLAA), 374–402 aa region of CD2v Ct harboring the di-Leu motif in one (CT2) or four copies (CT2x4) and the 240–304 aa region that does not harbor the di-Leu motif (CTS). (B): COS cells were lysed and incubated with immobilized GST-CD2v-WT, GST-CD2v-LLAA, GST-CT2, GST-CT2x4, GST-CTS or GST alone. They were co-precipitated using glutathione-Sepharose beads, separated by 10% SDS-PAGE followed by immunoblotting with an anti-AP-1 monoclonal antibody. Densitometry values of anti-AP-1 bands relative to anti-GST are presented in the graph below. A representative experiment of at least three independent experiments is shown.