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An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the approximately 30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.

The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.

An RNA replicase of GA phage, one of the Group II RNA phages, was isolated and purified to a homogeneous state. By SDS polyacrylamide gel analysis, the purified GA replicase was found to contain four different subunits, numbered I, II, III, and IV, the molecular weights of which were 74,000, 60,000, 47,000, and 36,000, respectively. Three of them, I, III, and IV, proved to be host-coded proteins, ribosomal protein S1 (I), and elongation factors Tu (III) and Ts (IV) of protein biosynthesis, respectively. On a phosphocellulose column, the RNA replicase was separated into two components: One composed of subunits I and II, and the other composed of subunits III and IV. Each component alone had no replicase activity. However, when the two components were combined at 0 degree C, 60% of the replicase activity was restored within 10 min. The purified GA replicase catalyzed the GA phage RNA-directed synthesis of template-size RNA. However, the maximum level of product RNA synthesized was less than 20% of the amount of template RNA added. RNA-RNA hybridization experiments indicated that the product RNA included only the RNA strand complementary to the template RNA, and not the viral strand.

A certain factor(s) derived from Escherichia coli was found to extensively stimulate RNA synthesis by the RNA replicase of phage GA. This factor(s), named GA-HF (host factor(s) for GA RNA replication), was partially purified from an uninfected cell extract and characterized. In the presence of GA-HF, GA replicase synthesized 50-100 times more RNA than was synthesized in its absence, and was capable of synthesizing both the viral strand as well as its complementary strand. This factor(s) could not be replaced by HFI, which is necessary for the replication of Q beta RNA by Q beta replicase. In the presence of GA-HF, the GA RNA replication system has a characteristic template specificity. Group I and II phage RNAs, but none of the Group III and IV phage RNAs, showed template activity.

Phage SP RNA-dependent RNA polymerase (SP replicase) was purified from Escherichia coli infected with RNA phage SP. The enzyme was found to be composed of four non-identical polypeptides, i.e. subunits I, II, III, and IV and molecular weights of 74,000, 69,000, 47,000, and 36,000 daltons, respectively. As in the case of phage Qbeta replicase, the largest polypeptide is identical with the ribosomal protein S1, and subunits III and IV with polypeptide chain elongation factors EF-Tu and EF-ts, respectively.. This is based on the behaviour of the subunits on SDS-polyacrylamide gel electrophoresis, isoelectric focusing and immunological cross-reaction. Subunits I, III, and IV of SP replicase are derived from the host cell, while subunit II is coded by phage RNA genome. The striking coincidence of the composition and entity of the structural components of SP replicase with those of Qbeta replicase may indicate the structural and functional requirements of host-derived polypeptides in RNA replicase. The binding activity of S1 (in 70S ribosome comples) to poly (U) is retained in SP replicase complex. In contrast, the GDP binding activity of EF-Tu is masked in SP replicase. It is concluded that S1 is required functionally whereas EF-Tu.EF-Ts are required structurally in RNA replicase.

Amino acid sequences of 2 giant non-structural polyproteins (F1 and F2) of infectious bronchitis virus (IBV), a member of Coronaviridae, were compared, by computer-assisted methods, to sequences of a number of other positive strand RNA viral and cellular proteins. By this approach, juxtaposed putative RNA-dependent RNA polymerase, nucleic acid binding ("finger"-like) and RNA helicase domains were identified in F2. Together, these domains might constitute the core of the protein complex involved in the primer-dependent transcription, replication and recombination of coronaviruses. In F1, two cysteine protease-like domains and a growth factor-like one were revealed. One of the putative proteases of IBV is similar to 3C proteases of picornaviruses and related enzymes of como- nepo- and potyviruses. Search of IBV F1 and F2 sequences for sites similar to those cleaved by the latter proteases and intercomparison of the surrounding sequence stretches revealed 13 dipeptides Q/S(G) which are probably cleaved by the coronavirus 3C-like protease. Based on these observations, a partial tentative scheme for the functional organization and expression strategy of the non-structural polyproteins of IBV was proposed. It implies that, despite the general similarity to other positive strand RNA viruses, and particularly to potyviruses, coronaviruses possess a number of unique structural and functional features.