Conserved motifs within Ebola and Marburg virus VP40 proteins are important for stability, localization, and subsequent budding of virus-like particles

Abstract

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the (96)LPLGVA(101) sequence of eVP40 and the corresponding (84)LPLGIM(89) sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the (96)LPLGVA(101) motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the (96)LPLGVA(101) motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-DeltaLPLGVA failed to rescue the budding defective eVP40-DeltaLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-DeltaLPLGIM successfully rescued budding of mVP40-DeltaLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

FIG. 1.

Diagram of conserved motifs in the matrix proteins of negative-sense RNA viruses. Alignment of the partial matrix protein sequences of Ebola virus (EBOV), Marburg virus (MARV), Hendra virus (HeV), Nipah virus (NiV), measles virus (MeV), Newcastle disease virus (NDV), Sendai virus (SeV), simian virus 5 (SV5), Tupaia paramyxovirus (TPMV), and human parainfluenza virus type 1 (HPV-1), showing conservation of the motif sequence of interest (boxed residues in boldface). The numbers represent the amino acid positions within the matrix proteins beginning from their N termini.

FIG. 2.

Budding assay for WT and mutant eVP40 and mVP40 proteins. (A) Schematic representation of eVP40-WT, eVP40-Δ₉₆LPLGVA₁₀₁, eVP40-Δ₇PT/PY₁₃, mVP40-WT, and mVP40-Δ₈₄LPLGIM₈₉. The amino acid positions of the deleted motifs are indicated. (B) Human 293T cells were transfected with eVP40-WT, eVP40-ΔLPLGVA, eVP40-ΔPT/PY, mVP40-WT, and mVP40-ΔLPLGIM. Western blot with anti-eVP40 and anti-Flag antiserum to detect eVP40 and mVP40 proteins, respectively, in cell extract and VLP samples.

FIG. 3.

Intracellular localization of eVP40 and mVP40 WT and mutant proteins. (A) Human 293T cells were transfected with the indicated eVP40 plasmids. Transfected cells were stained with Alexa 594 (red) and DAPI (blue) and examined by confocal microscopy. Cells transfected with pCAGGS vector were used as a negative control. (B) Human 293T cells were transfected with the indicated mVP40 plasmids. pCAGGS vector served as a negative control. Transfected cells were stained and then examined by confocal microscopy.

FIG. 4.

Budding assay for eVP40 WT and point mutants. (A) Schematic representation of eVP40-WT, L96A, P97A, and L98A. (B) Human 293T cells were transfected with the indicated plasmids. Western blotting with anti-eVP40 antiserum was used to detect the indicated protein in cell extract and VLP samples.

FIG. 5.

Intracellular localization of eVP40-WT and point mutants. Plasmids encoding eVP40-WT and the indicated point mutants were transfected into 293T cells, and protein expression was visualized by using confocal microscopy.

FIG. 6.

Gel filtration analysis of eVP40 and mVP40 proteins. Human 293T cells were transfected with the indicated plasmids. Cells were lysed with PBS containing 1% Triton X-100. Cell lysates were separated on a FPLC column. (A) The chromatogram for the eVP40-WT elution is shown as absorbance (280 nm) versus elution volume. Molecular mass standards (670, 158, 44, 17, and 1.35 kDa) were plotted along the dashed line as log values versus elution volume. (B) Western blot analyses of each protein are shown. pCAGGS empty vector served as a negative control.

FIG. 7.

Pulse-chase analysis of eVP40 and mVP40 proteins. Human 293T cells were transfected with eVP40-WT, eVP40-ΔLPLGVA, mVP40-WT, and mVP40-ΔLPLGIM. Transfected cells were metabolically labeled with [³⁵S]methionine-cysteine for 30 min and then chased for the indicated times. Cell lysates were immunoprecipitated with anti-eVP40 or anti-Flag antisera to detect eVP40 or mVP40 proteins. Immunoprecipitated proteins were separated by SDS-PAGE, and proteins were visualized by autoradiography.

FIG. 8.

Rescue of VLP budding. (A) Human 293T cells were transfected with the indicated plasmids, and both VLPs and cell extracts were harvested at 36 h posttransfection. Western blotting was performed to detect eVP40-ΔLPLGVA in VLPs and cell extracts using anti-eVP40 antiserum. An expression control for Ebola virus GP is shown. (B) Human 293T cells were transfected with the indicated plasmids, and both VLPs and cell extracts were harvested at 36 h posttransfection. Western blotting was performed to detect eVP40-ΔLPLGVA in VLPs and cell extracts. An expression control for mVP40-WT is shown. (C) Human 293T cells were transfected with the indicated plasmids, and both VLPs and cell extracts were harvested at 36 h posttransfection. Western blotting was performed to detect mVP40-ΔLPLGIM in VLPs and cell extracts using anti-Flag antiserum. An expression control for eVP40-WT is shown. Coexpression of eVP40-WT (1.0 μg) was able to rescue budding of mVP40-ΔLPLGIM into VLPs to levels comparable to those of mVP40-WT alone (compare lanes 6 and 7). (D) Human 293T cells were transfected with the indicated plasmids. Western blotting was performed to detect the eVP40-ΔLPLGIM-Flag protein in VLPs and cell extracts using anti-Flag antibody. eVP40-WT was detected in cell extracts using anti-eVP40 antiserum. An expression control for eVP40-ΔLPLGIM-Flag is indicated. (E) Human 293T cells were transfected with the indicated plasmids. Expression of mVP40-Flag protein in VLPs and cell extracts was detected by using anti-Flag antiserum. Expression of mVP40-WT-HA fusion protein was detected by using anti-HA antiserum. An expression control for mVP40-ΔLPLGIM-Flag is shown.