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

Abstract

Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.

Fig. 1.

Z is a direct inhibitor of L catalytic activity. (A) Cellular-based replicon assay for MACV RNA synthesis where functional viral polymerase results in the production of gaussia luciferase. Luciferase activity was measured from the supernatant of cells expressing pMCm-GLuc replicon in the presence of L (L) or a catalytically inactivated mutant (L-SDD). Error bars represent the SD from the mean of at least three independent experiments. (B and C) Replicon assay as in A with cells transfected with the viral replicon and L alone (−) or with MACV Z (Z) or GST-tagged MACV Z (Z^GST) plasmids. Luciferase values were normalized to cells expressing only the replicon plasmid and wild-type L. Error bars represent the SD as in A and conditions that are statistically significant from the wild-type L control are designated with the symbol * (P < 0.001 or for Z^GST P = 0.0016). (D) MACV in vitro RNA synthesis. Purified L (L: WT) or a catalytically inactivated mutant (L: SDD) was incubated with a wild-type template (template RNA: WT), a 3′ terminal dideoxy template (template RNA: dd) or an RNA template with a mutated promoter binding site (template RNA: Mut) in the presence of GpC primer as described in the text. RNA products were labeled with [α-³²P]-UTP and separated by denaturing gel electrophoresis. (E and F) RNA products generated from RNA synthesis reactions supplemented with purified GST-tagged MACV Z (+), MBP-tagged MACV Z (MBP-Z), untagged MACV Z (Z), or free MBP (MBP) and analyzed as in D. (G and H) Total RNA synthesis was quantified with a PhosphorImager and graphed as values normalized to reactions containing wild-type L and RNA template. Error bars represent the SD from the mean of at least three independent experiments (*P < 0.001). See also Figs. S1 and S2.

Fig. 2.

Viral RNA synthesis regulation by Z is species specific. (A) In-cell replicon assay as in Fig. 1 with cells expressing an empty cassette (Vec), MACV Z (Z^M), JUNV Z (Z^J), or LCMV Z (Z^L). (B) RNA products resulting from in vitro RNA synthesis reactions supplemented with GpC primer and purified GST-MACV Z (Z^M) or GST-LCMV (Z^L) were analyzed and (C) quantified as in Fig. 1 (*P < 0.001). (D) In vitro RNA synthesis reactions supplemented with GpC primer and an increasing amount of untagged MACV Z (Z^M), GST-MACV Z (GST-Z^M), or GST-LCMV (GST-Z^L). RNA products were analyzed and (E) quantified as in Fig. 1. Untagged MACV Z and GST-MACV Z data were analyzed with the GraphPad software package. Error bars for each point correspond to the SD from the mean of independent experiments.

Fig. 3.

Z and L form a direct heterodimeric complex. (A) GST pull-down assay using purified components. Purified MACV L (L), free GST (GST), GST-tagged MACV Z (Z^M), or GST-tagged LCMV Z (Z^L) were incubated in the designated combinations before purification with glutathione resin. Pelleted resin was washed three times and bound proteins were eluted and analyzed by 12% SDS/PAGE and colloidal Coomassie stain. Input proteins were loaded as markers and indicate one-third of the total input protein from each assay. (B) The molecular weight of the L–Z complex was estimated by preincubating L and MBP-tagged Z and comparing the gel-filtration elution profile of the complexed proteins with purified MACV L (L), MBP-tagged MACV Z (MBP-Z), and known molecular weight standards. See also Figs. S3 and S4.

Fig. 4.

Z blocks an early step of RNA synthesis initiation. (A) Gel shift assay of complex formation between L, RNA, and Z. Radiolabeled 3′ RNA was incubated with buffer alone (−) or the indicated combinations of purified L (L), GST-tagged MACV Z (Z^M), and GST-tagged LCMV Z (Z^L). The resulting complexes were separated by nondenaturing gel electrophoresis. (B) In vitro RNA synthesis reactions in the absence of GpC primer were labeled with [α-³²P]-GTP or (C) 5′ end-labeled [α-³²P]-GpC and supplemented with untagged MACV Z (Z^M). (D) Analysis of MACV in vitro transcripts subsequently labeled after RNA synthesis. Transcripts from unlabeled RNA synthesis reactions in the absence of GpC primer were purified by phenol-chloroform extraction and ethanol precipitation and then subsequently labeled by capping the 5′ end with vaccinia virus guanyltransferase and [α-³²P]-GTP and separated as in B. (E) RNA products were analyzed and quantified as in Fig. 1 (*P < 0.001 or for [α-³²P]-GpC P = 0.0042). See also Fig. S5.

Fig. 5.

Model of arenavirus RNA synthesis regulation by Z. Low concentrations of Z permit ongoing RNA synthesis, whereas high concentrations of Z result in an inhibited Z–L–RNA complex bound to the viral promoter. As described in the text, the Z–L–RNA complex may serve as an important intermediate ensuring L is packaged into mature virions.