Kissing Interaction between 3′ Noncoding and Coding Sequences Is Essential for Porcine Arterivirus RNA Replication

329

435

The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3 NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3 NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3 end of the HCV genome that is essential for replication. The hepatitis C virus (HCV) is the only member of theHepacivirinae within the Flaviviridae family (46). These viruses possess an RNA genome of positive polarity that in the case of HCV has a length of about 9,600 nucleotides (nt). Its genome carries a single long open reading frame (ORF) that is flanked at either end by highly conserved nontranslated regions (NTRs) (2, 37). The 5Ј NTR harbors an internal ribosome entry site (IRES) directing the synthesis of the viral polyprotein which is cleaved into 10 different products. RNA replication takes place in the cytoplasm in a distinct compartment (12) and requires viral proteins NS3 to NS5B as well as host cell factors (28,43). In addition, cisacting replication elements (CREs) are important for the synthesis of RNA progeny. Such CREs may reside at various positions in an RNA genome but are most often found in the NTRs. In the case of HCV, the minimal signals that are essential for RNA replication have been mapped within the NTRs. The 5Ј NTR is composed of four distinct domains, with domains 1 and 2 being sufficient for RNA replication (10, 19). The 3Ј NTR has a tripartite structure and is composed of a highly variable region immediately downstream of the stop codon of the polyprotein, a polypyrimidine [poly(U/UC)] tract of variable length, and a highly conserved 98-nt-long RNA element designated the X-tail (22,41,42,52). The latter has the potential to form three stemloop (SL) structures, of which the most 3Ј terminal, designated SL1, has been confirmed experimentally (5,15). In contrast, the structures of the two other SLs that are located upstream are much less clear (5,15,56). Both the X-tai...

Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

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

Friebe

Boudet

Simorre

et al. 2005

329

435

“…These interactions can be mediated by base pairing or by proteins. In porcine reproductive and respiratory syndrome virus, a kissing interaction between stem-loop structures in the ORF and the 3Ј NTR is required for RNA replication (63). Other examples of longdistance RNA-RNA interactions important for RNA virus replication have been found for flaviviruses (31,72), flock house virus (39), RNA coliphage Q␤ (34), and barley yellow dwarf luteovirus (20).…”

Section: Discussionmentioning

confidence: 99%

A cis -Acting Replication Element in the Sequence Encoding the NS5B RNA-Dependent RNA Polymerase Is Required for Hepatitis C Virus RNA Replication

You

Stump

Branch

et al. 2004

203

299

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5 and 3 ends of the RNA genome should provide new insights into HCV RNA replication.The first molecular clones of hepatitis C virus (HCV) were reported in 1989 (11). Comparative sequence analysis revealed that HCV is related to flavi-and pestiviruses (12), and HCV was subsequently placed in the family Flaviviridae. The genomic RNA of viruses in this family has a long open reading frame (ORF) flanked by nontranslated regions (NTRs) at the 5Ј and 3Ј termini (48). HCV initiates translation of the ORF via an internal ribosome entry site (IRES) (25, 51). Translation of the ORF yields a polyprotein that is cleaved co-and posttranslationally by host and viral proteases. The HCV polyprotein gives rise to 10 viral proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3). NS5B, the viral RNAdependent RNA polymerase, comprises the C-terminal portion of the polyprotein. The 5Ј and 3Ј NTRs of HCV have cis-acting replication elements (CREs) which are essential for the viral life cycle (14,32,33,36,70) and are known to bind cellular proteins (15,21,26,46,57,67).Coding regions of viral RNAs often have embedded nucleic acid signals and overlapping reading frames that encode proteins, as in X174 (52), hepatitis B virus (16), and Rous sarcoma virus (27). Embedded RNA signals include promoters (29), nucleation sites for encapsidation (2,30,35,49), and other types of CREs (5,18,40,43,44,53). Among the best characterized is the picornavirus CRE (17,18,40,43,45,71). The picornavirus CRE acts as a template for the uridylylation of VPg, the protein primer for genome replication, and thus plays a direct role in initiation of RNA replication. The exac...

“…These domains, which are highly conserved among PRRSV isolates, may play a critical role in virus replication. The mutations introduced by primer F15301 (Table 1) are both situated in this 3Ј nontranslated region stem-loop (36). Even though none of the mutations are situated within the sevennucleotide sequence essential for the kissing interaction, mutations anywhere in this domain might weaken the interaction, thereby reducing viral replication.…”

Section: Discussionmentioning

confidence: 99%

Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Nielsen

Liu

Nielsen³

et al. 2003

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK 12 . To rescue infectious virus, capped RNA was transcribed in vitro from the pOK 12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5 end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.