Analysis of the Cell Type-Dependence on the Arenavirus Z-Mediated Virus-Like Particle Production

Abstract

Several arenaviruses are highly pathogenic to humans, causing hemorrhagic fever. Discovery of anti-arenavirus drug candidates is urgently needed, although the molecular basis of the host- and organ-specific pathogenicity remains to be fully elucidated. The arenavirus Z protein facilitates production of virus-like particles (VLPs), providing an established method to assess virus budding. In this study, we examined the efficiency of VLP production by solely expressing Z protein of several different arenaviruses. In addition, we analyzed the role of the late (L)-domain of the arenavirus Z protein, which is essential for the interaction with ESCRT proteins, in VLP production among different cell lines. VLP assay was performed using Z proteins of Junín virus (JUNV), Machupo virus (MACV), Tacaribe virus (TCRV), Latino virus (LATV), Pichinde virus (PICV), and Lassa virus (LASV) in six different cell lines: HEK293T, Huh-7, A549, Vero76, BHK-21, and NIH3T3 cells. JUNV, MACV, and LASV Z proteins efficiently produced VLPs in all tested cell lines, while the efficiencies of VLP production by the other arenavirus Z proteins were cell type-dependent. The contribution of the L-domain(s) within Z protein to VLP production also highly depended on the cell type. These results suggested that each arenavirus has its own particle-production mechanism, which is different among the cell types.

Keywords: L-domain; Z; arenavirus; cell type-dependence; virus-like particle.

FIGURE 1

Genome and structure of arenavirus. (A) The genome of arenaviruses consists of two segments of single-stranded ambisense RNA, S and L. Each segment encodes two viral proteins separated by an intergenic region (IGR), a non-coding region forming a hairpin structure. The S segment encodes viral nucleoprotein (NP) and glycoprotein precursor (GPC), whereas the L segment encodes viral RNA-dependent RNA polymerase (L) and the matrix protein (Z). (B) Structure of the arenavirus: GP1/2 (blue), L (purple), NP (yellow) and Z (green). The viral genome (red) is encapsidated by NP. (C) Z protein consists of three domains, N-terminus (orange), central RING domain (light blue) and C-terminus (bright green) containing the L-domain (red).

FIGURE 2

Schematic representation of L-domain within arenavirus Z proteins and the L-domain mutants used in this study. L-domains within Z proteins of the following arenaviruses. (A) Junín virus (JUNV), (B) Machupo virus (MACV), (C) Tacaribe virus (TCRV), (D) Latino virus (LATV), (E) Pichinde virus (PICV), (F) Lassa virus (LASV), and their respective alanine mutant forms. WT, wild-type; Mut, mutant.

FIGURE 3

Arenavirus Z-mediated VLP production. HEK293T cells (A), Huh-7 cells (B), A549 cells (C), Vero76 cells (D), BHK-21 cells (E), and NIH3T3 cells (F) were transfected with expression plasmids for JUNV, MACV, TCRV, LATV, PICV, or LASV Z-FLAG. At 48 h post-transfection (hpt) (A–C,E) or 72 hpt (D,F), VLPs and whole-cell lysates were collected and analyzed by western blot (WB). In all experiments, actin served as a loading control. VLP production by JUNV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Role of the PTAP motif in JUNV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for WT (PTAP) or mutant (Mut; PTAP → AAAA) JUNV Z-FLAG using TransIT LT1 transfection reagent. At 48 hpt, VLPs and whole-cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysates were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, actin served as a loading control. VLP production from JUNV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). WT, wild-type; Mut, mutant. ***p < 0.001.

FIGURE 5

Role of the PSAP motif in MACV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for the WT (PSAP) or mutant (Mut; PSAP → AAAA) MACV Z-FLAG using TransIT LT1 transfection reagent. At 48 hpt, VLPs and cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysates were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, actin served as a loading control. VLP production from MACV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). WT, wild-type; Mut, mutant. **p < 0.01, ***p < 0.001.

FIGURE 6

Role of the ASAP motif in TCRV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for the WT (ASAP) or mutant (Mut; ASAP → AAAA) TCRV Z-HA using TransIT LT1 transfection reagent. At 48 hpt, VLPs and cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysates were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, actin served as a loading control. VLP production from TCRV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). WT, wild-type; Mut, mutant. ***p < 0.001.

FIGURE 7

Role of the PTAP motif in LATV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for the WT (PTAP) or mutant (Mut; PTAP → AAAA) LATV Z-FLAG using TransIT LT1 transfection reagent. At 48 hpt, VLPs and cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysate were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, the actin served as a loading control. VLP production from LATV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). WT, wild-type; Mut, mutant. ***p < 0.001.

FIGURE 8

Role of the PSAPPY motif in PICV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for the WT (PSAPPY) or three mutants, PICV Z-Mut1 (PSAPPY → AAAAPY), PICV Z-Mut2 (PSAPPY → AAAPPY) and PICV Z-Mut3 (PSAPPY → PSAPPA), using TransIT LT1 transfection reagent. At 48 hpt, VLPs and cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysates were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, actin served as a loading control. VLP production from PICV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels). WT, wild-type; Mut, mutant. ***p < 0.001.

FIGURE 9

Role of the PTAP and PPPY motifs in LASV Z-mediated VLP production: (A) HEK293T cells were transfected with the expression plasmid for the WT (PTAP-PPPY), Mut1 (PTAP → AAAP), or Mut2 (PPPY → PPPA) LASV Z using TransIT LT1 transfection reagent. At 48 hpt, VLPs and cell lysates were collected and analyzed by western blot (WB). (B) Huh-7 cells were transfected and analyzed as described above. (C) A549 cells were transfected with the same expression plasmids using Lipofectamine 3000 transfection reagent and analyzed as described in panel (A). (D) Vero76 cells were transfected with the same expression plasmids as described in panel (A). At 72 hpt, VLPs and whole-cell lysates were collected and analyzed by WB. (E) BHK-21 cells were transfected and analyzed as described in panel (A). (F) NIH3T3 cells were transfected and analyzed as described in panel (D). In all performed experiments, actin served as a loading control. VLP production from LASV Z-WT was set at 1.0 as a standard, and the data shown are averages and standard deviations of four independent experiments (right panels) WT, wild-type; Mut, mutant. ***p < 0.001.