Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV) Chimera: Interaction between the Poliovirus Cloverleaf and a Sequence in the HCV 5′ Nontranslated Region Results in a Replication Phenotype

Zhao, Wei; Lahser, Frederick; Wimmer, Eckard

doi:10.1128/jvi.74.13.6223-6226.2000

Cited by 24 publications

(27 citation statements)

References 26 publications

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“…We found that expression of the luciferase gene was severely impaired in transfected cells when the full-length HCV 5Ј NTR was directly fused to the PV IRES, whereas no reduction of luc translation was observed with the other 5Ј variants (data not shown). This result indicated an interference between the HCV and the PV RNA elements similar to what was recently found with a recombinant PV that carries an HCV IRES to direct translation of the PV polyprotein (56).…”

Section: Vol 75 2001supporting

confidence: 58%

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Friebe

Lohmann

Krieger

et al. 2001

J Virol

290

239

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Sequences in the 5 and 3 termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5 end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5 NTR required for RNA replication. We show that deletions introduced into the 5 terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5 NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5 NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5 NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5 NTR of HCV RNA essential for replication of this virus. Hepatitis C virus (HCV) is a distinct member of the familyFlaviviridae, comprising a group of enveloped viruses to which the flaviviruses like Yellow fever virus and the pestiviruses Border disease virus, Classical swine fever virus (CSFV), and Bovine viral diarrhea virus (BVDV) belong (33). In the majority of cases, HCV causes a persistent infection that is frequently asymptomatic or associated with only mild symptoms. However, persistently infected patients are at high risk to develop a chronic liver disease that can lead to liver cirrhosis and eventually hepatocellular carcinoma (39). In the absence of efficient therapies, chronic hepatitis C has become the main indication for liver transplantation.HCV possesses a single-stranded RNA genome of positive polarity. This plus-strand RNA carries a single long open reading frame that encodes a polyprotein of ϳ3,010 amino acids. Mature viral proteins are generated by a series of proteolytic cleavages that are mediated by host cell signal peptidases and two viral proteinases (for a review, see reference 4). The structural proteins core, envelope protein 1 (E1), and E2 are located in the amino-terminal one-third of the polyprotein, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are located in the remainder. Of the latter, NS3 to NS5B are required and sufficient for RNA replication (28). NS3 possesses proteinase as well as nucleoside triphosphatase (NTPase) and helicase activities and NS5B is the RNA dependent RNA polymerase (RdRp) (3,6,17,23,26,49). The function of the hydrophobic polypept...

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Section: Vol 75 2001supporting

confidence: 58%

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Friebe

Lohmann

Krieger

et al. 2001

J Virol

290

239

View full text Add to dashboard Cite

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“…On the other hand, deletions up to nucleotides 27, 17, and 9 resulted in medium, small, and minute plaque sizes, respectively. The authors explained this phenomenon on the basis of a putative interaction between the HCV sequence up to nucleotide 31 and the poliovirus cloverleaf, which plays a key role in polioviral replication (Zhao et al 2000). However, on the basis of the present study, we suggest that this phenomenon may be, at least in part, attributed to long-range RNA-RNA interaction, which inhibits HCV Interaction between 5NTR and coding region of HCV www.rnajournal.org IRES-dependent translation, as the HCV sequence nucleotides 428-442 was present in the chimeric viruses.…”

Section: Discussionmentioning

confidence: 99%

“…Translation of the polyprotein is directed by an internal ribosomal entry site (IRES) spanning most of the 5Ј nontranslated region (5ЈNTR) in combination with a portion of the core-coding region that augments the IRES-dependent translation (Tsukiyama-Kohara et al 1992;Wang et al 1994Wang et al , 2000Rijnbrand et al 1995;Honda et al 1996a;Lu and Wimmer 1996;Hwang et al 1998;Zhao et al 2000). With the exception of small hairpin structures located within the most 5Ј segment of the 5ЈNTR (domain I), all of the predicted stem-loop structures are essential for the internal initiation directed by HCV IRES (Wang et al 1994(Wang et al , 1995Rijnbrand et al 1995Rijnbrand et al , 2001Honda et al 1996a,b;Kamoshita et al 1997;.…”

Section: Introductionmentioning

confidence: 99%

Long-range RNA–RNA interaction between the 5′ nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Kim

Lee

Kim

et al. 2003

RNA

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Hepatitis C virus (HCV) is a positive-sense RNA virus ∼9600 bases long. An internal ribosomal entry site (IRES) spans the 5 nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.

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“…Using this method, desired foreign antigens could be incorporated without size restriction; however, the recombinant virus is replication defective and requires a helper virus or capsid protein-expressing system for each cycle of propagation (12,30,34,35). The propagation-defective viral vector is attractive for its safety feature, even in the neuronal delivery of cytokine (8), but may also pose obstacles for inducing vigorous immune responses.The third strategy focused on construction of the dicistronic genome by placing a foreign insert together with the 5' noncoding internal ribosome entry site (1,2,24,27,45), but this vector strategy was flawed by frequent observation of the gradual deletion of the foreign gene in the early stages of serial passage. The cause of the genetic instability was not determined, but it was apparently related to the spatial restriction of the PV capsids.…”

mentioning

confidence: 99%

“…The third strategy focused on construction of the dicistronic genome by placing a foreign insert together with the 5' noncoding internal ribosome entry site (1,2,24,27,45), but this vector strategy was flawed by frequent observation of the gradual deletion of the foreign gene in the early stages of serial passage. The cause of the genetic instability was not determined, but it was apparently related to the spatial restriction of the PV capsids.…”

mentioning

confidence: 99%

Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System

Lee

Kim

Hyun

et al. 2002

J Virol

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Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.Many attempts have been made to manipulate poliovirus (PV) as a favorable vaccine vector because of its attractive characteristics of safe use, low cost, convenient administration, and long-lasting protective immunity in both mucosal and systemic immune responses, which have been established for decades. Four different strategies have been employed to date in the development of PV-based versatile vaccine vectors. Early efforts were devoted to attaching small epitopes to one of the neutralizing domains in the capsid protein (9, 10, 14, 17, 36). However, widespread application of this hybrid virion strategy was limited by the size of the epitope (smaller than 25 amino acids) that could be inserted. The second strategy was to replace one of the structural viral genes with the protein insert (8,9,12,30,34,35). Using this method, desired foreign antigens could be incorporated without size restriction; however, the recombinant virus is replication defective and requires a helper virus or capsid protein-expressing system for each cycle of propagation (12,30,34,35). The propagation-defective viral vector is attractive for its safety feature, even in the neuronal delivery of cytokine (8), but may also pos...

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Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV) Chimera: Interaction between the Poliovirus Cloverleaf and a Sequence in the HCV 5′ Nontranslated Region Results in a Replication Phenotype

Cited by 24 publications

References 26 publications

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Long-range RNA–RNA interaction between the 5′ nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System

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