Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

Abstract

West Nile virus (WNV), 1 a mosquito-borne member of Flaviviridae family that consists of more than 70 human pathogens, was first isolated in 1937 from the blood of a female patient in the West Nile district of Uganda (1). Since then, the virus has been isolated in humans, birds, and bird-feeding mosquitoes, Culex univittatus. Serological studies revealed a broad distribution of the virus in Africa, West Asia including India, and the Middle East, but only occasionally isolated in Europe, and is thought to be spread by migratory birds (Ref. 2, for reviews, seeRefs. 3 and 4). WNV belongs to the Japanese encephalitis serocomplex group, which includes St. Louis encephalitis, Murray Valley encephalitis, Kunjin virus, and Japanese encephalitis virus (5, 6). WNV was not previously isolated in the Western hemisphere. The first outbreak of WNV infections occurred in New York in 1999 (7-10) in which 62 people were confirmed to be infected, seven of which were fatal. Since then, transmission of WNV is spreading rapidly throughout the United States; 12 states along the eastern seaboard in 2000 to 25 states and the District of Columbia by 2001 (3).WNV, like other flaviviruses, contain a positive (ϩ)-strand RNA of ϳ11 kb in length that is capped at the 5Ј-end and nonpolyadenylated at the 3Ј-end. WNV RNA contains conserved sequence elements within the 5Ј-and 3Ј-terminal regions (TR), which include two self-complementary cyclization motifs (5Ј-CYC and 3Ј-CYC) of 9 nt in length (11). There is a highly conserved stem-loop structure within the 3Ј-terminal 96 nt of 3Ј-UTR of flaviviral RNAs and a less conserved stem-loop structure within 5Ј-UTR (12-14) (for reviews, see Refs. 4,15,and 16). In addition, there is a potential pseudoknot tertiary structure within the 3Ј-terminal stem-loop structure of WNV RNA (17). Several host proteins have been shown to bind to the 5Ј-and 3Ј-UTR although their functional role in viral RNA replication has not been clearly established (18 -21) (for a review, see Ref. 22). By mutational analysis, the 3Ј stem-loop region within the 3Ј-UTR was shown to be important for viral replication in vivo (23,24) and in vitro (25).The flaviviral RNA genome encodes a single polyprotein precursor that were processed co-translationally and posttranslationally into three structural proteins (capsid, C; precursor membrane, prM; its processed form, membrane protein, M; and the envelope, E) and at least seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (for reviews, see Refs. 4,15,and 16). NS3 is a multifunctional protein; the N-terminal region of NS3 contains the serine protease domain and it requires NS2B for cleavage at the junctions of 2A-2B, 2B-3, 3-4A, and 4B-5. NS3 contains conserved motifs found in RNA helicases of the DEXH family (for reviews, see Refs. 4,15,and 16). Purified NS3 was shown to possess NTPase and RNA helicase activities (26 -32) as well as 5Ј-RNA triphosphatase activity (33, 34), the first enzyme required in 5Ј-cap synthesis.NS5 is the largest of the flaviviral nonstructural pro...

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

“…It has been previously proposed that the 5Ј-3Ј hybridized complementary sequences present in the DV genome constituted the promoter for RNA synthesis (Ackermann and Padmanabhan 2001; You et al 2001;Nomaguchi et al 2004;Alvarez et al 2005b). Here, we demonstrated that these sequences do not promote viral RNA synthesis per se and that the RNA element responsible for this activity is the SLA structure.…”

“…1C). We used a well-characterized assay in which templatelength RNA products of negative polarity were demonstrated for DV RdRp activity (Ackermann and Padmanabhan 2001;Nomaguchi et al 2004). We found that substitutions of specific nucleotides within SLB or 5ЈCS yielded RNAs that were very active as templates (MSLB and MCS RNAs).…”

“…It has been previously proposed that interactions between the 5Ј and 3Ј ends of the flavivirus genome could constitute the structure recognized by the polymerase during minus-strand RNA synthesis (Ackermann and Padmanabhan 2001;You et al 2001;Nomaguchi et al 2004;Alvarez et al 2005b). In addition, it has been reported that RNA elements at the 5Ј end of DV genome, besides the CS sequence, enhance in vitro polymerase activity (You et al 2001).…”

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

“…NS5 comprises an N-terminal methyltransferase (MTase) domain that catalyses both guanine N-7 and ribose 29-O methylations during viral RNA cap formation (Zhou et al, 2007) and a C-terminal domain that harbours the RNA-dependent RNA polymerase (RdRp) (Tan et al, 1996;Guyatt et al, 2001;Nomaguchi et al, 2004;Selisko et al, 2006). The crystal structure of the WNV RdRp revealed classic RdRp structure bearing palm, thumb and finger subdomains (Malet et al, 2007).…”

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

“…Additionally, there are two pairs of conserved sequences (59CS1, 39CS1, 59UAR and 39UAR) that together induce DENV cyclization (Alvarez et al, 2005a, b; Hahn et al, 1987). These motifs are essential for negativestrand RNA synthesis of DENV (Ackermann & Padmanabhan, 2001; Alvarez et al, 2005a, b;Villordo & Gamarnik, 2009;You & Padmanabhan, 1999) and other flaviviruses (Bredenbeek et al, 2003;Corver et al, 2003;Jones et al, 2005;Khromykh et al, 2001;Lo et al, 2003;Nomaguchi et al, 2004). On the other hand, sequences present within a large stem-loop structure located at the 59 end as well as a conserved oligo(U) track function as the promoter for viral polymerase activity (Lodeiro et al, 2009).…”