RING Finger Z Protein of Lymphocytic Choriomeningitis Virus (LCMV) Inhibits Transcription and RNA Replication of an LCMV S-Segment Minigenome

Abstract

Arenaviruses have a bisegmented negative-strand RNA genome whose proteomic capability is limited to only four polypeptides, namely, nucleoprotein (NP), surface glycoprotein (GP) that is proteolytically processed into GP1؉GP2, polymerase (L), and a small (11-kDa) RING finger protein (Z). The role of Z during the Lymphocytic choriomeningitis virus (LCMV) life cycle is poorly understood. We investigated the function of Z in virus transcription and replication by using a reverse genetic system for the prototypic arenavirus LCMV. This system involves an LCMV minigenome and the minimal viral trans-acting factors (NP and L), expressed from separated cotransfected plasmids. Cotransfection of the Z cDNA strongly inhibited LCMV minigenome expression. The effect required synthesis of Z protein; its magnitude was dose dependent and occurred with levels of Z protein substantially lower than those observed in LCMV-infected cells. Coexpression of Z did not prevent the encapsidation of plasmid supplied minigenome, but it affected both transcription and RNA replication similarly. Mutations in Z that unfolded its RING finger domain eliminated its inhibitory activity, but RING proteins not related to Z did not affect LCMV minigenome expression. Consistent with the minigenome results, cells transiently expressing Z exhibited decreased susceptibility to infection with LCMV. Lymphocytic choriomeningitis virus (LCMV)is the prototypic member of the family Arenaviridae, which includes important human pathogens such as Lassa fever and Junin viruses (11,41,43,53,55). In addition, LCMV provides one of the most widely used model systems to study viral persistence and pathogenesis (7,10,37,38,39,40,45).The LCMV genome consists of two negative-sense singlestranded RNA segments, L and S, with approximate sizes of 7.2 and 3.4 kb, respectively (44,48,52,53). Each RNA segment has an ambisense coding strategy, encoding two proteins in opposite orientations, separated by an intergenic region (IGR) (48, 53). The S RNA directs synthesis of the three major structural proteins: the nucleoprotein, NP (ca. 63 kDa) (44,48,53), and the two virion glycoproteins, GP-1 (40 to 46 kDa) and GP-2 (35 kDa), which are derived by posttranslational cleavage of a precursor polypeptide, GP-C (75 kDa) (48,53,54,60). Tetramers of GP-1 and GP-2 make up the spikes on the virion envelope and mediate virus interaction with host cell surface receptor (6, 13). The L RNA segment encodes a high-molecular-mass protein (L, ca. 200 kDa) (52), which has the characteristic motifs conserved in all of the viral RNA-dependent RNA polymerases (46), and a small (11-kDa) RING finger protein (Z) (50). The NP, the most abundant viral protein in virus-infected cells, is associated with the viral RNA to form the nucleocapsid (NC), which is the template for the viral RNA polymerase (23,24). The L protein is thought to be the main viral component of the arenavirus polymerase (23,48,53). The NC associated with the viral polymerase constitutes the viral ribonucleoprotein (RNP), which is active in ...

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

“…1C). Previous results have revealed a similar phenotype using overexpressed Z in other arenaviral replicon systems (10,11,19), yet it has been impossible to determine whether Z directly impacts the viral RNA synthesis machinery or functions through alteration of an essential host cellular process. We therefore established an in vitro system to examine a potential direct effect of Z on viral RNA synthesis.…”

“…All of the enzymatic machinery required for viral RNA synthesis and mRNA transcription is contained within L, a 250-kDa multifunctional protein that is organized as a central ring-like RNA-dependent RNA-polymerase (RdRP) domain with three accessory appendages (9). As ectopic overexpression of Z induces potent inhibition of replicon-based reporter protein expression (10,11), Z has been hypothesized as an important regulator of L and arenaviral gene expression. However, Z is known to interact with several host factor binding partners, and it remains unclear whether Z directly regulates the viral RNA synthesis machinery or acts through alteration of an essential host process.…”

Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein

“…The cells were infected 2 h later with LCMV (moi ϭ 2). Sixty hours later, cell lysates were prepared and assayed for CAT activity as described (12). …”

“…Detailed PCR conditions are available from the authors on request. Northern blots were prepared and hybridized with appropriate 32 P-double-strand DNA probes as described (12). Cloning of plasmid pSr(Ϫ) is described in Fig.…”

“…Moreover, we found that the viral 5Ј and 3Ј UTR, together with the intergenic region, are sufficient cis-acting signals for RNA synthesis by the LCMV RNA-dependent RNA polymerase. Z was not required for MG transcription and replication and exhibited a dose-dependent inhibitory effect on both processes (12). We have also shown that assembly and budding of LCMV infectious virus-like particles (VLPs) require both GP and Z (10).…”