Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A - PubMed (original) (raw)

Comparative Study

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A

Takashi Irie et al. J Virol. 2004 Mar.

Erratum in

Abstract

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.

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Figures

FIG. 1.

FIG. 1.

Budding assay of VSV M proteins. (A) Schematic representation of expression plasmids encoding Mwt and chimeric M40. The amino acid sequences of the L-domains are indicated by their single-letter code. (B) Radiolabeled cell lysates and VLPs were immunoprecipitated with anti-M MAb, and immunoprecipitates were analyzed by SDS-PAGE. (C) M proteins in VLPs were quantitated by densitometry with the ImageJ 1.24 software, and the bars represent an average of six independent experiments. The level of Mwt was normalized to 1.0, and the relative value of M40 is indicated.

FIG. 2.

FIG. 2.

Effect of DN VPS4A on budding of VP40-WT and VP40-dPTA VLPs. Human 293T cells were cotransfected with a combination of VP40-WT or VP40-dPTA and the indicated VPS4A plasmid (wt or DN EQ mutant). Cell lysates (A) and VLPs (B) were immunoprecipitated with the appropriate antibodies to detect VP40 or VPS4A as indicated. VP40 in VLPs was quantitated by PhosphorImager analysis.

FIG. 3.

FIG. 3.

Effect of DN VPS4A on budding of VSV Mwt and M40 VLPs. BHK-21 cells were infected with VvT7 and then cotransfected with the indicated plasmids. Cell lysates (A) and VLPs (B) were immunoprecipitated with the appropriate antibodies to detect VSV M or VPS4A. (C) Mwt and M40 present in VLPs were quantitated, and the bars represent an average of three independent experiments. The level of Mwt in VLP samples from cells not receiving VPS4A (−) was normalized to 1.0.

FIG. 4.

FIG. 4.

Effect of DN VPS4A on VSV budding. (A and B) COS-1 cells were transfected with vector alone (−), VPS4-WT (wt), or VPS4-EQ (EQ). At 24 h p.t., the cells were infected with VSV at an MOI of 0.1 PFU/ml, and culture medium was harvested at 0, 4, 6, 8, 10, and 12 h p.i. Virions (A) and cell lysates (B) were harvested at 12 h p.i. M protein was immunoprecipitated from virions (A), and both M and VPS4A proteins (B) were immunoprecipitated from cell extracts. (C) The virus titer was determined by plaque assay, and titers were plotted against time p.i.

FIG. 5.

FIG. 5.

Growth kinetics of recombinant RV expressing VPS4A proteins. (A) Schematic representation of the genome of WT SPBN and recombinant genomes containing VPS4-WT, VPS4-EQ, and VPS4-KQ. All inserts and 5′ and 3′ promoter regions were sequenced. (B) BSR-T7 cells were infected with the indicated viruses at an MOI of 0.01 PFU/ml, and culture medium was harvested at 8, 24, 48, and 72 h p.i. Virus titer at each time point was determined in duplicate on BSR cells and is plotted against time p.i.

FIG. 6.

FIG. 6.

Effect of TSG101 depletion on budding of Mwt and M40 VLPs. (A) Human 293T cells were transfected with TSG101-specific or nonspecific (NS) siRNAs together with a TSG101-encoding plasmid (pCAGGS.MCS vector). TSG101 was detected by immunoprecipitation with anti-TSG101 antibody. (B) Immunoprecipitation of Mwt and M40 proteins from cell extracts (lanes 1 to 6) and VLPs (lanes 7 to 12) in the absence of siRNA, in the presence of NS siRNA, or in the presence of TSG101 siRNA as indicated. (C) Quantitation of Mwt and M40 in VLPs from three independent experiments. The level of Mwt protein in VLP samples from NS siRNA-transfected cells was normalized to 1.0.

FIG. 7.

FIG. 7.

Effect of TSG101 depletion on VSV budding. Human 293T cells were transfected with TSG101-specific or NS siRNAs as indicated and then infected with VSV at an MOI of 0.1 PFU/ml. M protein present in cell lysates and virions was detected by immunoprecipitation. Virus production (10 h p.i.) from cells receiving TSG101-specific or NS siRNA was determined by a duplicate plaque assay on BHK-21 cells.

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