Genetic recombination of poliovirus in a cell-free system

Duggal, Rohit; Cuconati, Andrea; Gromeier, Matthias; Wimmer, Eckard

doi:10.1073/pnas.94.25.13786

Cited by 46 publications

(56 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, the frequency of such recombination events is predicted to be far below detection within the timeframe and reporter expression levels of our system. By analogy with other positivestranded RNA viruses, intertypic recombination rates, e.g., between the type 1 replicon and the type 2 helper BVDV, are predicted to occur with a frequency of ϳ10 Ϫ8 (12,17,43,57). Thus, RNA recombination seems unlikely as the mechanism yielding the luciferase expression observed in our studies.…”

Section: Discussionmentioning

confidence: 60%

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Ansari

Chen

Liang

et al. 2004

J Virol

View full text Add to dashboard Cite

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-toproline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.Virion assembly and release from the host cell constitute the final stages of the viral life cycle. These events are as important for viral pathogenesis in the host as the widely studied mechanisms of virus entry or genome replication. Enveloped virion assembly represents the convergence of viral proteins, nucleic acid, and host lipids to build an infectious particle. The process of virus budding, which characterizes enveloped viruses, is difficult to study because it involves a living cell. No cell-free virus budding systems have been developed to dissect this process.Hepatitis C virus (HCV) is an important human pathogen that causes persistent infections leading to chronic hepatitis, which can result in hepatocellular carcinoma (1, 2). To avoid the late sequelae of chronic HCV infections, millions of infected individuals await the development of effective antivirals (29). Efforts to explore all the possible drug targets for HCV, including virion assembly, have been hampered by the lack of robust in vitro cell culture systems that yield infectious virions (60). Much of our current knowledge on HCV virion assembly is derived from the study of virus-like particles (VLPs). Numerous reports support the concept that viral structural proteins carry enough structural information to direct the as...

show abstract

Section: Discussionmentioning

confidence: 60%

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Ansari

Chen

Liang

et al. 2004

J Virol

View full text Add to dashboard Cite

show abstract

“…For instance, characterization of the recombination sites within the poliovirus genome at nonphysiological and physiological conditions revealed that severe restrictions were applied to recombination within the P1 region at physiological conditions. Using an in vitro system for studying recombination in the poliovirus genome, Duggal et al (1997) observed fewer limitations of recombination between poliovirus genomes in vitro than in vivo. This suggests that recombination may occur outside eventual hot spots of recombination.…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported that the genetic lineage represented by the prototype strain of echovirus 9 (EV9H) is, as expected, closely related to the Barty genotype of EV9 (EV9B) in the structural genes while the non-structural genes of the P3 region are closely related to a lineage represented by the echovirus 18 prototype (EV18M) (Andersson et al, 2002). A recombination site was estimated by a maximum-likelihood approach to the 2C gene, one region that is a frequent recombination spot in polioviruses (Duggal et al, 1997;Duggal & Wimmer, 1999). However, the impact of recombination on the biology and evolution of enteroviruses has not been seriously assessed.…”

Section: Introductionmentioning

confidence: 99%

Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species

Lindberg¹,

Andersson²,

Savolainen

et al. 2003

Journal of General Virology

132

View full text Add to dashboard Cite

Enteroviruses show a high degree of sequence variation both between and within serotypes due to the lack of proofreading of the viral RNA-dependent RNA polymerase. In addition, recombination is known to occur not only within but also between different serotypes. We have previously shown that capsid coding sequences of coxsackievirus B4 (CVB4) cluster in several coexisting genotypes (intergenotypic nucleotide difference of 12 % or more) whereas a single lineage of echovirus 30 (EV30) has been prevailing and evolving throughout the last two decades. In the major capsid gene, VP1, clustering of both nucleotide and amino acid sequences correlates with serotype. We have now determined a 501 nucleotide sequence in the non-structural 3CD region of CVB4 and EV30 field strains. Phylogenetic analysis revealed that sequences of Human enterovirus B (HEV-B) were segregated in the 3CD region into three distinct clusters without the VP1-associated serotype/genotype correlation. One of the clusters comprised the E2 strain of CVB4, the EV30 prototype and five other CVB4 field strains whereas the other two clusters, in addition to CVB4 and EV30 strains, also included other HEV-B serotypes. We believe that intertypic recombination is the most likely explanation for the observed incongruence. Similarity analysis based on complete genomes of the CVB4 and EV30 prototypes and the CVB4 E2 strain revealed that a putative recombination spot was mapped within the 2B gene. The incongruence observed in the two genomic domains (P1 and P3) suggests a certain degree of independent evolution, which may be explained by interserotypic recombination within an enterovirus species. It is thus difficult to exclude recombination in the history of any given strain.

show abstract

“…The ability to observe copy-choice recombination between poliovirus genomes in a cell-free system has been demonstrated recently by Kirkegaard (46) and Wimmer (47). Notwithstanding, these experiments utilized a HeLa cell extract, and all of the poliovirus proteins were present.…”

Section: Template Switching Is the Primary Pathway For Formation Of Pmentioning

confidence: 99%

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

Arnold

Cameron

1999

Journal of Biological Chemistry

109

105

View full text Add to dashboard Cite

We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D pol . Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D pol -catalyzed nucleotide incorporation and RNA recombination are discussed.Positive-strand RNA viruses cause a variety of diseases in humans ranging from the common cold (1) to chronic hepatitis (2). Critical to the replication of the genomes of these viruses is the virus-encoded, RNA-dependent RNA polymerase (RdRP) 1 (3). As this enzymatic activity is unique to virus-infected cells, the viral RdRP represents a very attractive target for the design of antiviral agents to treat RNA virus infection. Most positive-strand RNA viruses are thought to have the same general replication strategy, and this belief is generally extended to include the mechanism of genome replication (3). RdRPs from many viruses have been characterized to some extent (3-9). However, in no instance is detailed, mechanistic information available. This fact is even true for viruses, such as poliovirus, for which genetic and biochemical systems to study genome replication have existed for many years (10).Notwithstanding, poliovirus is one of the best understood systems with respect to the biochemistry of genome replication and is, therefore, an invaluable paradigm for all positivestrand RNA viruses (10). Replication of poliovirus genome initiates within the poly(A)-tract at the 3Ј-end of genomic RNA from a complex comprising viral factors (3AB, 3CD, 3B, and 3D pol ) (11-13) and possibly cellular factors (14). 3AB and 3CD are required primarily to establish an initiation complex, possibly by recruiting the polymerase, 3D pol (13). 3AB is a RNAbinding protein capable of interacting with 3D pol (15,16) that possibly stimulates the rate of elongation of nascent RNA (17) or enhances the efficiency of primer utilization (18)....

show abstract

Genetic recombination of poliovirus in a cell-free system

Cited by 46 publications

References 26 publications

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

Contact Info

Product

Resources

About