Cells expressing the RING finger Z protein are resistant to arenavirus infection

Abstract

Arenaviruses include Lassa fever virus (LFV) and the South American hemorrhagic fever viruses. These viruses cause severe human disease, and they pose a threat as agents of bioterrorism. Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides: nucleoprotein (NP); surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Our investigators have previously shown that Z has a strong inhibitory activity on RNA synthesis mediated by the polymerase of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). In this report we show that cells transduced with a replication-deficient recombinant adenovirus expressing Z (rAd-Z) are resistant to LCMV and LFV infection. Virus cell entry mediated by LCMV or LFV GP was not affected in rAd-Z-transduced cells, but both virus transcription and replication were strongly and specifically inhibited, which resulted in a dramatic reduction in production of infectious virus. These findings open new avenues for developing antiviral strategies to combat the highly pathogenic human arenaviruses, including LFV.

FIG. 1.

Vero cells transduced with rAd-Z are specifically resistant to LCMV infection. (A) Expression levels of rAd-supplied Z protein depended on the MOI of rAd-Z. Cells were transduced with rAd-Z at various MOIs. Twenty hours later, cell lysates were prepared and analyzed by Western blotting using antibodies to Z or actin. (B and C) Vero cells were transduced with either rAd-Z or rAd-T7, both at an MOI of 2, and subsequently infected with either LCMV or MV (both at an MOI of 2). LCMV antigens were not detected in rAd-Z-transduced cells (B). At 48 h postinfection (p.i.), cells were fixed and analyzed by IF using antibodies to LCMV and MV. Production of infectious LCMV particles was dramatically and specifically reduced in rAd-Z-transduced cells (C). At the indicated time p.i., tissue culture supernatants were collected and titers of LCMV and MV were determined by plaque assay.

FIG. 2.

Vero cells expressing a Myc-tagged LFV Z (LFVZmyc) are specifically resistant to LCMV and LFV infection. (A) Expression levels of rAd-supplied LFVZ depended on the MOI of rAd-LFVZ. Cells were transduced with rAd-LFVZ at the indicated MOI. Twenty hours later, cell lysates were prepared and analyzed by Western blotting using antibodies to Myc and actin. (B and C) Vero cells were transduced with either rAd-LFVZ or rAd-T7, both at an MOI of 2, and subsequently infected with either LCMV (MOI of 2), MV (MOI of 2), or LFV (MOI of 2). At 48 h postinfection (p.i), cells were fixed and analyzed by IF with antibodies to LCMV, MV, or LFV. LCMV (B) and LFV (C) antigens were not detected in rAd-LFVZ-transduced cells. (D) Expression of LFV GP2 was dramatically reduced in both whole-cell extracts and tissue culture supernatants of rAd-LFV-transduced cells. Vero cells were transduced with either rAd-T7 or rAd-LFVZ (MOI of 2) and were subsequently infected with LFV (MOI of 2). Seven days after LFV infection, supernatants and whole-cell lysates were analyzed by Western blotting by using an antibody to LFV GP2.

FIG. 3.

(A) Cell entry mediated by LCMV or LFV GP is not affected in rAdZ-transduced cells. Vero cells were transduced with either rAd-T7 or rAdZ (both at an MOI of 2) and subsequently infected with VSVΔG pseudotyped with either VSV G or LCMV GP. Twelve hours later, cells were fixed and analyzed by IF using an antibody to the nucleoprotein of VSV. (B) Low levels of apoptosis induced by rAd-supplied Z do not affect LCMV replication. (i) Levels of apoptosis (as a percentage) were normalized with respect to those seen in STS (50 μM)-treated cells. (ii) STS (50 μM)-treated and untreated Vero cells were infected with LCMV (MOI of 1). At 24 h postinfection (p.i.), total cellular RNA was isolated and analyzed by Northern blot hybridization using an LCMV NP probe. (C) LCMV RNA synthesis, both transcription and replication, is inhibited in cells transduced with rAdZ. Cells were transduced with either rAd-Z or rAd-GFP or were nontransduced, and subsequently cells were infected with LCMV. At the indicated time p.i., total cell RNA was isolated and analyzed by Northern blot hybridization using a [³²P]NP double-stranded DNA probe that hybridizes to both NP mRNA (transcription) and S RNA (replication).