Efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein

Abstract

The Z protein has been shown for several arenaviruses to serve as the viral matrix protein. As such, Z provides the principal force for the budding of virus particles and is capable of forming virus-like particles (VLPs) when expressed alone. For most arenaviruses, this activity has been shown to be linked to the presence of proline-rich late-domain motifs in the C terminus; however, for the New World arenavirus Tacaribe virus (TCRV), no such motif exists within Z. It was recently demonstrated that while TCRV Z is still capable of functioning as a matrix protein to induce the formation of VLPs, neither its ASAP motif, which replaces a canonical PT/SAP motif in related viruses, nor its YxxL motif is involved in budding, leading to the suggestion that TCRV uses a novel budding mechanism. Here we show that in comparison to its closest relative, Junin virus (JUNV), TCRV Z buds only weakly when expressed in isolation. While this budding activity is independent of the ASAP or YxxL motif, it is significantly enhanced by coexpression with the nucleoprotein (NP), an effect not seen with JUNV Z. Interestingly, both the ASAP and YxxL motifs of Z appear to be critical for the recruitment of NP into VLPs, as well as for the enhancement of TCRV Z-mediated budding. While it is known that TCRV budding remains dependent on the endosomal sorting complex required for transport, our findings provide further evidence that TCRV uses a budding mechanism distinct from that of other known arenaviruses and suggest an essential role for NP in this process.

FIG. 1.

Expression and VLP release by JUNV and TCRV proteins. (A) Schematic representation of the linear structure of the Z proteins of the Old World arenaviruses LCMV and LASV, as well as the clade B New World arenaviruses, including JUNV and TCRV. The relative positions of known and putative late-domain motifs, as well as the late-domain-like ASAP motif of TCRV, are illustrated. (B) Budding of JUNV NP, GP, and Z. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-JUNV Z (C-Flag), pCAGGS-JUNV NP (N-Flag), or pCAGGS-JUNV GPC (C-Flag). After 48 h, cell lysates were collected and analyzed by SDS-PAGE and Western blotting. Cell supernatants were purified over a 20% sucrose cushion before also being analyzed by SDS-PAGE and Western blotting. All proteins were detected using a mouse anti-Flag M2 monoclonal antibody diluted 1:500 and an Alexa Fluor 680-coupled goat anti-mouse secondary antibody diluted 1:5,000. (C) Budding of TCRV NP, GP, and Z. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-TCRV Z (C-Flag), pCAGGS-TCRV NP (N-Flag), or pCAGGS-TCRV GPC (C-Flag). The lysates and supernatants were harvested and analyzed as described for panel B. (D) Quantification of JUNV and TCRV protein release. Following Western blotting, protein bands for both supernatants and cell lysates were quantified using an Odyssey Imager and the protein release was calculated as the amount of protein released in the total supernatant as a percentage of the total protein in the supernatant and cell lysate fractions. The values to the left of panels B and C are molecular sizes in kilodaltons.

FIG. 2.

Incorporation of other viral components into Z VLPs. (A) Incorporation of JUNV NP and GPC into JUNV Z-induced VLPs. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-JUNV Z (C-Flag) alone or together with 1 μg of pCAGGS-JUNV NP (N-Flag), pCAGGS-JUNV GPC (C-Flag), or both. Differences in the absolute plasmid masses transfected were compensated for by the addition of empty pCAGGS vector. After 48 h, cell lysates were collected and analyzed by SDS-PAGE and Western blotting (lower panel). Cell supernatants were purified over a 20% sucrose cushion before also being analyzed by SDS-PAGE and Western blotting (upper panel). All proteins were detected using a mouse anti-Flag M2 monoclonal antibody diluted 1:500 and an Alexa Fluor 680-coupled goat anti-mouse secondary antibody diluted 1:5,000. (B) Incorporation of TCRV NP and GPC into TCRV Z-induced VLPs. Experiments were carried out using pCAGGS-TCRV NP (N-Flag), pCAGGS-TCRV GPC (C-Flag), and pCAGGS-TCRV Z (C-Flag) as described above. (C and D) Quantification of JUNV and TCRV protein incorporation into VLPs. Following Western blotting, protein bands for both supernatants and cell lysates were quantified using an Odyssey Imager and the protein release was calculated as the amount of protein released in the total supernatant as a percentage of the total protein in the supernatant and cell lysate fractions.

FIG. 3.

Organization of JUNV and TCRV Z, NP, and GP inside multicomponent VLPs. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-JUNV Z (C-Flag) alone or together with 1 μg of pCAGGS-JUNV NP (N-Flag), pCAGGS-JUNV GPC (C-Flag), or both (upper panel). Alternatively cells were transfected with 1 μg of pCAGGS-TCRV Z (C-Flag) alone or together with 1 μg of pCAGGS-TCRV NP (N-Flag), pCAGGS-TCRV GPC (C-Flag), or both (lower panel). Differences in the absolute plasmid mass transfected were compensated for by the addition of empty pCAGGS vector. After 48 h, cell supernatants were purified over a 20% sucrose cushion before being resuspended in PBS alone, PBS plus protease K (Prot K), or PBS plus Triton X-100 (TX-100) plus protease K. Samples were incubated at 37°C for 1 h before being analyzed by SDS-PAGE and Western blotting. All proteins were detected using a mouse anti-Flag M2 monoclonal antibody diluted 1:500 and an Alexa Fluor 680-coupled goat anti-mouse secondary antibody diluted 1:5,000.

FIG. 4.

Enhancement of Z VLP budding in the presence of NP. (A) Release of TCRV and JUNV Z in the presence or absence of NP. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-JUNV Z (C-Flag) and 1 μg of either pCAGGS-JUNV NP (N-Flag) or pCAGGS vector. Similarly, cells were also transfected with pCAGGS-TCRV Z (C-Flag) and 1 μg of either pCAGGS-TCRV NP (N-Flag) or pCAGGS vector. After 48 h, cell lysates were collected and analyzed by SDS-PAGE and Western blotting. Cell supernatants were purified over a 20% sucrose cushion before also being analyzed by SDS-PAGE and Western blotting. All proteins were detected using a mouse anti-Flag M2 monoclonal antibody diluted 1:500 and an Alexa Fluor 680-coupled goat anti-mouse secondary antibody diluted 1:5,000. (B) Kinetics of JUNV and TCRV protein release. Samples were transfected as described for panel A, and samples were harvested at 24 h, 48 h, 72 h, and 96 h posttransfection. Following Western blotting, protein bands for both supernatants and cell lysates were quantified using an Odyssey Imager and the protein release was calculated as the amount of protein released in the total supernatant as a percentage of the total protein in the supernatant and cell lysate fractions.

FIG. 5.

Effects of mutations in putative late-domain motifs on TCRV Z budding, enhancement of budding by NP, and NP incorporation into VLPs. (A) Schematic representation of the of TCRV Z mutants analyzed. The relative positions of putative late-domain motifs, as well as the late-domain-like ASAP motif of TCRV, are shown, and the mutations made in these motifs are indicated. (B) Budding of TCRV Z late-domain mutants. 293 cells were transfected with 1 μg of eukaryotic expression plasmid pCAGGS-TCRV Z (C-Flag) (wild-type [Wt]) or the various mutants indicated in panel A together with either 1 μg of empty pCAGGS vector or 1 μg of pCAGGS-NP (N-Flag). After 48 h, cell lysates were collected and analyzed by SDS-PAGE and Western blotting. Cell supernatants were purified over a 20% sucrose cushion before also being analyzed by SDS-PAGE and Western blotting. All proteins were detected using a mouse anti-Flag M2 monoclonal antibody diluted 1:500 and an Alexa Fluor 680-coupled goat anti-mouse secondary antibody diluted 1:5,000. Subsequently, bands for both supernatants and cell lysates were quantified for Z release using an Odyssey Imager and the protein release was calculated as the amount of protein released in the total supernatant as a percentage of the total protein in the supernatant and cell lysate fractions. (C) Samples prepared as described for panel B were also quantified with respect to the release of NP in VLPs as described for panel B.