Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome

Abstract

Point mutations that resulted in a substitution of the conserved 3-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of 6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3-penultimate cytidines and a 3-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.The Flaviviridae comprise three genera of positive-strand RNA viruses (Flavivirus, Pestivirus, and Hepacivirus). Their infectious genomes have a single open reading frame and no 3Ј-poly(A) tail (17). The 5Ј-and 3Ј-penultimate and/or -terminal nucleotides of genomic RNA are conserved within each genus, and they are each adjacent to one or more conserved terminal stem-loops: e.g., for Hepacivirus RNA (4, 18, 37), Flavivirus RNA (8,36,39,41,43,48), and Pestivirus RNA (3, 13, 53). Other common features are the similarities in gene order and function of the major viral enzymes in NS3 (protease and helicase) and in NS5/NS5B (RNA-dependent RNA polymerase [RdRp]) (17, 30).The Flavivirus RNA replication strategy has been established with Kunjin virus (KUN) (11,12,24,(49)(50)(51). The genomic or RNA positive strand is translated, a replication complex is formed cotranslationally on the 3Ј untranslated region (UTR) of the template RNA, and it transcribes an RNA negative strand that remains base paired with the RNA positive strand as a replicative form (RF). We found no evidence for the presence of a free RNA negative strand in KUN replicontransfected cells (22). We have defined the consensus composition of the KUN replication complex (RC) as NS1, NS3, NS5, and hydrophobic nonstructural proteins NS2A and NS4A plus the RF (24,34,50,51). The RF functions as a recycling template (shown by the kinetics of pulse-chase labeling) for asymmetric and semiconservative replication of progeny RNA positive strands by the viral replicase, and no subgenomic RNA is synthesized (11). Purified 32 P-labeled KUN RF was converted to a replicative intermediate (RI) when presented as a substrate to a lysate of KUN-infected cells (2). The Pestivirus Bovine viral diarrhea virus (BVDV) (15,47) and Hepacivirus Hepatitis ...

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

“…The multiple functions of the 3Ј-SL in viral replication and the pleiotropic effect of the mutations introduced could explain our observations. In this regard, the results are in agreement with previous studies that have shown an essential function of the last nucleotides of flavivirus genomes (25,26,41). Therefore, to further understand the complex process of the structural changes around the 3Ј-SL during viral replication in infected cells, more information about the multiple functions of the 3Ј-SL is necessary.…”

“…24), the mechanism by which this element controls RNA synthesis is still unclear. Specific bulges within the 3Ј-SL as well as nucleotides at the loop and the 3Ј terminal sequence were found to be essential for RNA replication (25)(26)(27)(28). The 5Ј bottom part of the large stem of the 3Ј-SL contains the 3Ј-UAR sequence, complementary to the 5Ј-UAR region of the 5Ј-UTR (12,13,21).…”

Kissing-Loop Interaction in the 3′ End of the Hepatitis C Virus Genome Essential for RNA Replication

“…29). The 3Ј-terminal ϳ95 nt of these viruses form a conserved SL structure that in the upper loop carries the highly conserved pentanucleotide motif CACAG that at least for Kunjin virus was shown to be crucial for RNA replication (18). Given this high conservation between different flaviviruses, we assume that cellular proteins that are expressed in various tissues and conserved between different species are involved in the formation of a replication complex at the 3Ј end of these genomes.…”

Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

“…This hypothesis is based on in vitro transcription studies using RdRp from bacteriophage Q␤, Turnip yellow mosaic virus, and TCV (67,68). Studies on flaviviruses and bromoviruses have also indicated that some or all of the 3Ј-terminal nucleotides are critical for replication in vivo and transcription in vitro (3,25,37,(49)(50)(51), although these nucleotides may not be important for stable interaction between the RdRp and the template (9). Despite the ability of TCV RdRp to initiate opposite the penultimate C in a sequence containing CCA repeats (68), the 3ЈCCC on the natural satC template is dispensable for basic transcription in vitro (20).…”