Identification of a Novel Function of the AlphavirusCapping Apparatus

Abstract

Semliki Forest virus (SFV)1 is member of the Alphavirus genus of the Togaviridae family. SFV has a positive-stranded 42 S RNA genome of 11.5 kilobases. RNA replication of SFV takes place in the cytoplasm and is catalyzed by the viral RNA-dependent RNA polymerase, which contains the virusspecific proteins Nsp1-4. These are cleavage products of a large (2342 aa) nonstructural polyprotein P1234. In the RNA polymerase complex all Nsps are in close association with each other (1, 2). The parental 42 S RNA is copied to complementary minus-strands, which in turn are used as templates for the synthesis of new 42 S RNA plus-strands and subgenomic 26 S mRNAs. During plus-strand synthesis, the 5Ј ends of the 42 S and 26 S RNAs become modified with covalently attached m 7 GpppA (the cap0 structure) (3-5). Capping of the RNAs is believed to be obligatory also for the replication of Alphavirus, since a point mutation specifically destroying the guanylyltransferase activity of Nsp1 is lethal for the virus (6).The functions of Alphavirus Nsps in replication have been studied using various genetic and biochemical approaches (1, 2). Nsp4 is the catalytic component of the RNA polymerase (7,8), whereas the functions of the phosphoprotein Nsp3 (9) are poorly defined (10, 11). Nsp2 has several distinct functions. It has nucleoside triphosphatase (NTPase) activity at its aminoterminal half (12), which is vital for the virus replication (13). Nsp2 has RNA helicase activity, which utilizes NTP hydrolysis as the energy source (14). The carboxyl-terminal part of the protein is a papain-like protease responsible for the autocatalytic cleavages of the nonstructural polyprotein (2, 15, 16). The carboxyl-terminal part has a nuclear localization sequence, which is responsible for sequestering of about half of the molecules to the nucleus during infection (17, 18). Furthermore, Nsp2 regulates transcription of the subgenomic 26 S RNA (Ref.19 and references therein). Here we show that Nsp2 has yet an additional activity required for capping of the virus mRNAs.Capping of cellular mRNAs occurs in the nucleus and comprises four different reactions. RNA 5Ј-triphosphatase removes the ␥-phosphate from the 5Ј end of the nascent RNA molecule (pppRNA 3 ppRNA). Guanylyltransferase reacts with a GTP molecule to form a covalent complex with GMP, which is then transferred from guanylyltransferase to the 5Ј end of RNA to form G(5Ј)ppp(5Ј)NpRNA. Methylation by guanine-7N-methyltransferase yields an RNA molecule with the cap0 structure (m 7 GpppNpRNA). Further methylation by nucleoside-2Ј-Omethyltransferase of the riboses of the penultimate and the adjacent nucleotides yields cap1 and cap2 structures, respectively (20,21).Unlike cellular mRNAs, the capping of Alphavirus RNAs takes place in the cytoplasm and is carried out by reactions that differ from the nuclear reactions as follows. (i) Nsp1 catalyzes transfer of the methyl group from S-adenosylmethionine to GTP to yield m 7 GTP (methyltransferase reaction), and (ii) a covalent guanylate complex Nsp1-m 7 GMP i...

Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein

“…1A, lanes 1-5), whereas P12 ∧ 3 ∧ 4 yielded nsP1 and P234 (lanes 6 -10). In these experiments nsP1 was seen as a double band, due to aberrant initiation at a downstream methionine ( [11][12][13][14][15]. Interestingly, polyproteins containing the noncleavable 1/2 site were not processed at the 2/3 site either (Fig.…”

Site-specific Protease Activity of the Carboxyl-terminal Domain of Semliki Forest Virus Replicase Protein nsP2

“…Protein Expression and Purification-Recombinant proteins were expressed in E. coli BL21(DE3) (Stratagene) and purified using Ni 2ϩ -affinity chromatography as described previously (16,15). Protein purification was monitored by 10 -17% SDS-PAGE (25), and protein concentration was determined with the Bradford assay (26) using Bio-Rad reagents.…”

“…nsP2 Expression Plasmids-Full-length nsP2 of SFV was produced and purified as described previously (15). To obtain a set of nsP2 mutants, containing progressive amino-terminal truncations, nine separate PCR amplifications were carried out using Pfu Turbo DNA polymerase (Stratagene), SFV infectious cDNA as a template and 3Ј-Mut (Table I) as common 3Ј primer.…”

“…The amino-terminal domain of SFV nsP2 (residues 1-470) has been shown to house several enzymatic activities including RNA triphosphatase (15), nucleoside triphosphatase (16), and RNA helicase (17). Interestingly, a significant amount of nsP2, synthesized during infection, is transported into the nucleus (18,19).…”

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

“…Viruses achieve this by encoding their own capping enzyme apparatus such as the vaccinia virus (VACV) D1/D12 subunit, baculovirus LEF-4 and reovirus l2 protein (Gross & Shuman, 1998a;Luongo et al, 2000;Shuman, 1990). In viruses such as reovirus and baculovirus, the same enzyme (l2 and LEF-4, respectively) catalyses both RTPase and mRNA GT activities, whereas VACV, West Nile virus and alpha virus encode separate RTPases (Benzaghou et al, 2006;Myette & Niles, 1996;Vasiljeva et al, 2000).…”