Vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate RNA synthesis and reveal mutations that affect mRNA cap methylation.

Vesicular stomatitis virus (VSV), the prototypic Rhabdovirus, has a nonsegmented negative-sense (nsNS) RNA genome of 11,161 nucleotides (nt). Infection is initiated by delivery into the host cell cytoplasm of a transcription-competent viral core (46), comprising the genomic RNA encapsidated by the nucleocapsid (N) protein and associated with the viral polymerase, a complex of the phosphoprotein (P) and the large polymerase (L) subunit (17). The genomic RNA is copied by the input polymerase to yield five capped and methylated mRNAs that encode the viral N, P, matrix (M), glycoprotein (G), and L proteins. Our current understanding of mRNA synthesis in VSV is summarized as follows. In response to a specific promoter element (32, 49, 50), the polymerase initiates synthesis at the start sequence of the first gene (11, 51) to produce the N mRNA which is modified at its 5′ terminus to yield the cap structure 7mGpppAmpApCpApGpApUpApUpC (2). In response to a conserved gene-end sequence the polymerase polyadenylates and terminates the N mRNA (8). Termination at the end of the N gene is essential for polymerase to initiate synthesis at the start of the next gene, to produce the P mRNA (1, 5). The mRNAs are not synthesized in equimolar quantities (48); rather, their abundance decreases with the distance between the gene and the 3′ promoter. This gradient of transcription reflects a poorly understood transcriptional attenuation event that is localized to the gene junction regions (27). The 241-kDa L protein contains the active site for ribonucleotide polymerization (44) and is responsible for cotranscriptional formation of the 5′-mRNA cap structure (21-23, 30, 31) and 3′-poly(A) tail (25). Although the total number of the genes can vary, this strategy for gene expression is shared by all viruses in the families Rhabdoviridae, Filoviridae, and Paramyxoviridae. The 5′-mRNA cap structure is formed by a series of enzymatic reactions, each of which are distinct to those employed by the host. Specifically, for VSV the 5′ pppApApCpApG must be modified to remove two phosphates to yield a 5′ pApApCpApG which is capped by transfer of GDP to yield the GpppApApCpApG cap structure (2). This contrasts with cellular capping in which an RNA triphosphatase removes a single phosphate and an RNA guanylyltransferase transfers GMP. The mRNA cap structure is then methylated by methyltransferases (MTases) at both guanine-N-7 (G-N-7) and ribose-2′-O (2′-O) positions to yield the 7mGpppAmpApCpApG cap structure (33). For VSV the two MTase activities use a single binding site for the methyl donor S-adenosyl-l-methionine (SAM) (31). In contrast, host mRNA cap structures are methylated by two separate enzymes, first at the G-N-7 position and then at the 2′-O position. Although best characterized for VSV, the unusual capping reactions are conserved among the nsNS RNA viruses, with the notable exception that some Paramyxovirus family members produce mRNAs that lack a 2′-O methyl group (12). Capping enzymes of many viruses are attractive candidates for chemotherapeutic intervention, and the methylation enzymes are no exception (13). In this regard, many adenosine analogues have been shown to inhibit the replication of a range of viruses in cell culture and diminish pathogenesis in small animal models (15). A proposed mechanism of inhibition mediated by such adenosine analogues is through interference with the host enzyme S-adenosyl homocysteine (SAH) hydrolase. SAH hydrolase is critical for converting SAH, the by-product of SAM-dependent MTases, into homocysteine and adenosine, and the products of this reaction are inhibitory to SAH hydrolase. Adenosine analogues such as 3-deazaeplanocin-A are potent antiviral agents, and previous work has shown a correlation between the ability of such analogues to inhibit SAH hydrolase and VSV replication in cell culture (14). Sinefungin (SIN) is a natural adenosine analog produced by Streptomyces griseolus and a known potent inhibitor of MTases. SIN is structurally related to SAM, except the methyl group that is donated from SAM is replaced by an amino group in SIN. Crystal structures of several MTases have been solved in complex with SIN (42, 43, 52), which binds to a region that overlaps the SAM binding site. SIN has been shown to have both antiviral and antifungal properties (18, 35, 37, 41, 52). Specifically, SIN was shown to inhibit the MTases of Newcastle disease virus (NDV) and vaccinia virus in vitro, as well as vaccinia virus plaque formation on L cells (41). While these experiments demonstrated that viral cap methylation reactions are inhibited by SIN, resistant mutants were not isolated. In addition, NDV mRNAs are not 2′-O methylated (12); thus, the effects of SIN on both cap methylations have not been previously described for an nsNS RNA virus. In this report, we show that SIN inhibits the VSV MTases in vitro, demonstrating that the viral L protein can serve as a direct target of SIN inhibition. We also show that SIN inhibits viral growth in cell culture, and we further isolate SIN-resistant (SINR) mutants. These mutants increased RNA synthesis in the presence of SIN, suggesting that upregulation of viral gene expression can lead to resistance. Sequence analysis of the genomes of resistant mutants identified previously unrecognized regions of the L gene that can impact mRNA cap methylation. These studies thus show that the inhibition of cap methylation enzymes of VSV can be used as a strategy to inhibit viral growth, and they suggest a means by which the virus can become resistant to such inhibition.