Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity

Abstract

The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (⌬97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that ⌬97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3 end of an acceptor RNA in the presence of divalent cations. Furthermore, ⌬97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. ⌬97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ϳ45% of that of the wild type in the presence of Mg 2؉ . Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn 2؉ . Identification of ⌬97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.Sindbis virus (SINV), the type species of the Alphavirus genus, is a plus-sense, single-stranded RNA virus. Its 11.7-kb genome is capped at the 5Ј end and polyadenylated at the 3Ј end (51). The 5Ј-terminal two-thirds of the genomic RNA encodes four nonstructural or replicase proteins. These nonstructural proteins are directly synthesized from the genomic RNA as two overlapping polyproteins, P123 and P1234. P1234 is produced by readthrough of a UGA termination codon present between the sequences encoding nsP3 and nsP4, which occurs 5 to 20% of the time during translation (30,50,51). The protease domain of nsP2 processes the polyproteins into various intermediates and individual nonstructural proteins (20). Nonstructural proteins together with an unknown host factor(s) constitute the alphavirus replication complex (51). The RNA-dependent RNA polymerase (RdRp), nsP4, which contains the signature GDD motif of viral RNA polymerases (24,25), is activated by cleavage from nascent P1234 to form an initial P123/nsP4 replication complex (47). This complex uses the genomic RNA as a template for the synthesis of minusstrand RNA. P123 is further processed at the 1/2 junction to produce an nsP1/P23/nsP4 complex, capable of synthesizing both minus-strand and 49S genomic RNAs. Finally, polyprotein P23 is cleaved at the 2/3 junction to produce a replication complex consisting of fully processed nonstructural proteins, which is responsible for the synthesis of 26S subgenomic mRNA ...

Genome 3′-end repair in dengue virus type 2

“…However, this mechanism has not been shown for RNA viruses that replicate in mammalian cells, and for the first time, this study describes the results of 39-end repair utilized by DENV2 polymerase. This intrinsic property of RdRPs has been suggested in in vitro experiments involving viral polymerases of DENV2 (Ackermann and Padmanabhan 2001), HCV (Ranjith-Kumar et al 2001), poliovirus (Arnold et al 1999), and Sindbis virus (Tomar et al 2006). In most cases, the nature of the nucleotides added by RdRPs was not random and seemed to be influenced by the 39-end nucleotides.…”

“…For example, 59-and 39-terminal SL structures conserved in many positive-strand RNA viral genomes provide a passive defense against nucleases with single-strand specificities. In addition, enzymatic mechanisms for the potential repair of damaged 39 ends have been suggested for various RNA viruses, including plant viruses such as brome mosaic virus and turnip crinkle virus (Rao et al 1989;Nagy et al 1997;Guan and Simon 2000), alphaviruses (Tomar et al 2006), poliovirus (Andrews et al 1985;Neufeld et al 1994), and hepatitis C virus (HCV) (Ranjith-Kumar et al 2001). However, except for studies in plant viruses, there have been no in vivo studies of 39-end repair in RNA viruses, including flaviviruses.…”

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

“…For example, one possibility is that the template generated by T7 RNA polymerase was restored to wt sequence by a cellular (or viral) terminal transferase activity. However, this seems unlikely since the 39 end of the template was created by a ribozyme and so contained a 29,39-cyclic phosphate at the terminus, rather than a 39-hydroxyl group (Tomar et al 2006). In addition, it would be surprising if a terminal transferase were to restore the length and sequence of the D2 template so efficiently that the product FIGURE 5.…”

“…As described in the Introduction, several RNA viruses use an RNA primer to initiate RNA replication (Garcin et al 1995;Paul et al 1998;Deng et al 2006;Tomar et al 2006;Ferrer-Orta et al 2009). If this were the case for RSV, it raises the question of how a sequence-specific primer could be generated.…”

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells

“…Within replication complexes, the polyproteins and individual nsPs have different functions in genomic and subgenomic plus-and minus-strand synthesis (Lemm et al, 1994(Lemm et al, , 1998Shirako & Strauss, 1994). nsP1 plays a role in capping virus RNAs (Mi et al, 1989;Scheidel et al, 1987), nsP2 is a multifunctional protein with helicase and 59 triphosphatase activities and is the protease that cleaves the non-structural polyprotein (Gomez de Cedró n et al, 1999;Ding & Schlesinger, 1989;Hardy & Strauss, 1989;Vasiljeva et al, 2000) and nsP4 is the RNA-dependent RNA polymerase and terminal adenyltransferase (Kamer & Argos, 1984;Tomar et al, 2006).…”