Architecture and regulation of negative-strand viral enzymatic machinery

Abstract

Negative-strand (NS) RNA viruses initiate infection with a unique polymerase complex that mediates both mRNA transcription and subsequent genomic RNA replication. For nearly all NS RNA viruses, distinct enzymatic domains catalyzing RNA polymerization and multiple steps of 5' mRNA cap formation are contained within a single large polymerase protein (L). While NS RNA viruses include a variety of emerging human and agricultural pathogens, the enzymatic machinery driving viral replication and gene expression remains poorly understood. Recent insights with Machupo virus and vesicular stomatitis virus have provided the first structural information of viral L proteins, and revealed how the various enzymatic domains are arranged into a conserved architecture shared by both segmented and nonsegmented NS RNA viruses. In vitro systems reconstituting RNA synthesis from purified components provide new tools to understand the viral replicative machinery, and demonstrate the arenavirus matrix protein regulates RNA synthesis by locking a polymerase-template complex. Inhibition of gene expression by the viral matrix protein is a distinctive feature also shared with influenza A virus and nonsegmented NS RNA viruses, possibly illuminating a conserved mechanism for coordination of viral transcription and polymerase packaging.

Figure 1. Structural architecture and organization of NS RNA viral polymerases. Conserved architecture and domain organization found in nonsegmented (top, purple) and segmented (bottom, orange) polymerases. The linear amino acid sequence of L and the tripartite influenza virus polymerase contain highly conserved regions dedicated to RNA synthesis (blue boxes). L and the influenza virus polymerase also contain blocks of conservation dedicated to 5′ cap formation (maroon boxes), including an endonuclease domain for cap-snatching (domain I or PA, segmented NS RNA viruses) or PRNTase / MTase domains for de novo cap synthesis (domains V and VI, nonsegmented NS RNA viruses). The regions containing cap formation enzymatic activities are also required for RNA synthesis, and the exact function of domains II and IV in the L protein of segmented NS RNA viruses remains unknown. EM of purified L from Machupo virus and vesicular stomatitis virus reveals a shared structural architecture conserved within NS RNA viral L proteins. L consists of a central ring-like RNA polymerase domain (blue highlight in cartoon) and a large appendage dedicated to 5′ cap formation (maroon highlight in cartoon) attached with a flexible linkage. As described in the text, comparison with EM analysis and 3D reconstruction of the influenza virus polymerase complex provides further insight into the architecture of L. Influenza A virus polymerase images reproduced with permission from refs. and

Figure 2. Inhibitory NS RNA viral proteins. Inhibition of gene expression by the viral matrix protein is a conserved feature of both segmented (top, orange) and nonsegmented (bottom, purple) NS RNA viruses. The table indicates the NS RNA virus families and main individual viruses for which inhibition by matrix or other accessory factors has been observed.

Figure 3. Model of arenavirus RNA synthesis regulation by the viral matrix protein. Low concentrations of matrix (Z) permit ongoing RNA synthesis, while 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. Other NS RNA viral matrix proteins or accessory inhibitory factors may play a similar role in viral replication, indicating a potentially conserved link between regulation of viral transcription and virion formation. (Fig. adapted from ref. 12).