Showing papers by "Sean P. J. Whelan published in 2009"

Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.

Abstract: Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein

Abstract: Nonsegmented negative-strand (NNS) RNA viruses use a common strategy to express their genomes (for a review, see reference 33). Their genomic RNA is encapsidated by the viral nucleocapsid (N) protein, and it is this N-RNA complex that serves as the template for the viral RNA-dependent RNA polymerase (RdRP). The RdRP comprises a virus-encoded large (L) polymerase protein that possesses all of the enzymatic activities necessary for the synthesis, capping, and polyadenylation of the mRNA and the replication of the genome. The L protein requires an additional cofactor, a phosphoprotein (P), which is required for template recognition. Some NNS RNA viruses require additional viral proteins for authentic RNA synthesis, but those proteins do not appear to contain catalytic activities. Vesicular stomatitis virus (VSV), the prototype of the family Rhabdoviridae, has long served as a model to understand RNA synthesis in the NNS RNA viruses. Purified VSV particles synthesize mRNA in vitro (3), and this can also be accomplished by using a recombinant N-RNA template purified from virus supplemented with recombinant L (rL) and rP (19, 22). During mRNA synthesis, the RdRP initiates synthesis at a 3′-proximal site to copy the viral genes in a polar and sequential manner (1, 2). In response to a specific promoter element, the RdRP initiates mRNA synthesis at a highly conserved gene start sequence, which for VSV is 3′-UUGUCNNUAG-5′ (21, 34, 35), and recognizes the cognate element in the nascent transcript to add an mRNA cap structure (23, 30, 31). Termination of mRNA synthesis is also controlled such that the VSV RdRP recognizes the sequence 3′-AUACUUUUUUU-5′ to polyadenylate the mRNA through reiterative transcription of the U tract and to terminate synthesis of the mRNA (4, 5, 14). The mechanism by which the VSV L protein adds the mRNA cap structure is distinct from that of all other known capping reactions. Specifically, a polyribonucelotidyltransferase (PRNTase) transfers a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate (23). This is in contrast to other capping reactions, in which a guanylyltransferase transfers GMP derived from GTP onto a diphosphate acceptor RNA (for a review, see reference 10). It is generally thought that the capping activity of L resides within one of six conserved regions (CR) that were identified by sequence alignments (26). Amino acid substitutions in a conserved GXXT(X68-70)HR motif in CRV prevent mRNA cap addition, implicating CRV as the PRNTase (19). In addition to the altered mechanism of cap addition, methylation of the cap structure is also unusual in that both guanine-N-7 (G-N-7) and ribose 2′-O positions are modified via what appears to be a single methyltransferase (MTase) domain within the L protein (11, 12, 17, 20). Sequence alignments and structural predictions between known 2′-O MTases and CRVI of L identified a putative MTase domain comprising the catalytic tetrad K-D-K-E and a GxGxG binding site for the methyl donor S-adenosyl-l-methionine (SAM) (6, 9). In transcription reactions carried out using rVSV, single amino acid substitutions in this K-D-K-E motif ablate all mRNA cap methylation (11, 12, 17), whereas single amino acid substitutions within the predicted SAM binding motif either prevented all cap methylation or specifically reduced G-N-7 methylation (20). These results suggested that the two methylase activities share a single binding site for SAM and that the two reactions can proceed in an unconventional order in which 2′-O methylation occurs first. However, those experiments could not determine whether the K-D-K-E motif is specifically required for G-N-7 methylation, as the lack of cap methylation may reflect a requirement for 2′-O methylation to occur first. In contrast to that idea, experiments with Sendai virus (SeV) demonstrated that purified rL protein or a C-terminal fragment comprising CRV and CRVI or CRVI alone was capable of G-N-7 methylation of virus-specific mRNA (24). However, those experiments did not detect a ribose 2′-O methylase activity associated with the SeV L protein and therefore could not address whether the two MTase activities reside within the same region of L. In the present study, we evaluated how the two MTase activities of the VSV L protein are regulated. We reconstituted methylation in vitro by using highly purified rL and capped RNA. This recapitulated both MTase activities independently of ongoing RNA transcription and allowed us to determine whether the methylase activities of L are coordinated. The results of this study demonstrate that the L protein functions as an efficient 2′-O MTase that facilitates a relatively inefficient G-N-7 MTase and that the K-D-K-E catalytic tetrad is essential for 2′-O methylation but also plays a role in G-N-7 methylation. We further demonstrate that the MTase activities are sequence specific and that they require a longer RNA substrate for methylation than does the PRNTase activity of L in vitro.

Opposing effects of inhibiting cap addition and cap methylation on polyadenylation during vesicular stomatitis virus mRNA synthesis.

Abstract: The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3′-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3′ termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5′ cap formation and 3′ polyadenylation.