Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity

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 45% 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 5 end and polyadenylated at the 3 end (51). The 5-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 and genomic RNA. The synthesis of minus-strand RNA ceases at about 4 h postinfection, while the plus-strand