Functional Analysis of Mosquito-Borne Flavivirus Conserved Sequence Elements within 3′ Untranslated Region of West Nile Virus by Use of a Reporting Replicon That Differentiates between Viral Translation and RNA Replication

Abstract

West Nile virus (WNV) belongs to the genus Flavivirus, which includes significant human pathogens such as dengue virus (DEN), yellow fever virus (YF), the tick-borne encephalitis virus complex, Japanese encephalitis virus (JE), Murray Valley encephalitis virus, and WNV. WNV is widely distributed throughout Africa, the Middle East, parts of Europe, Russia, India, Indonesia, and most recently, North America (6). Since the introduction of WNV into the United States in 1999, the virus has caused more than 4,156 known human cases and at least 284 deaths (for updates, see http://www.cdc.gov/ncidod /dvbid/westnile/surv&controlCaseCount03.htm). The WNV epidemic in the United States in 2002 represents the largest meningoencephalitis outbreak in the Western hemisphere and the largest WNV outbreak ever reported (7). Prevention and treatment of WNV infection have become public health priorities.Flavivirus virions contain a single plus-sense RNA genome of approximately 11 kb in length, which consists of a 5Ј untranslated region (UTR), a single long open reading frame (ORF), and a 3Ј UTR (24). The 5Ј and 3Ј UTRs are approximately 100 nt and 400 to 700 nt in length, respectively. The encoded polyprotein is cotranslationally and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (the glycoproteins NS1 and NS2a, the protease cofactor NS2b, the protease and helicase NS3, NS4a, and NS4b; and the methyltransferase and RNA-dependent RNA polymerase [RdRp] NS5) (8). During the flavivirus replication cycle, the synthesis of plus-and minus-sense RNAs is asymmetric; plus-sense RNA is produced in 10-to 100-fold excess over minus-sense RNA (11,23,28).The terminal nucleotides of the 3Ј UTR of flaviviruses can form highly conserved secondary and tertiary structures (4,5,24,26,31). Upstream of the 3Ј stem-loop structure, conserved sequences (CS) CS1, CS2, and repeated CS2 (RCS2) are retained among mosquito-borne flaviviruses. The JE subgroup and YF also have a distinct CS3 and repeated CS3 (RCS3). There is limited information available in regard to the functions of these CS elements. CS1 has the potential to base pair with a sequence within the conserved sequence in the 5Ј region of the capsid gene (5ЈCS) (13). Recent studies using DEN (33), Kunjin (16), and YF (10,22) replicons have shown that base pairing between 5ЈCS and CS1 is essential for flavivirus replication. For CS2, RCS2, CS3, and RCS3 elements, studies using

Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism

“…All of these viruses possess a 5Ј cap structure; however, the flavivirus reporters, like full-length flavivirus genomes, do not have 3Ј poly(A) tracts (12,30). Translation of each of the nonreplicating reporters was inhibited similarly to the SB-based reporter by IFN-␣/␤ priming of the PKR Ϫ/Ϫ MEFs (Fig.…”

Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus

“…In addition, the conserved sequences (CS) CS1, CS2, repeated CS2 (RCS2), CS3 and RCS3 exist upstream of the 39 SL structure of mosquito-borne flaviviruses (Lo et al, 2003). These SL structures and the CS region are important in genomic RNA synthesis (Lo et al, 2003;Shi et al, 1996;Tilgner & Shi, 2004).…”

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).…”