Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation

Machin, A.; Alonso, J.M.; Parra, Francisco

doi:10.1074/jbc.m100707200

Cited by 51 publications

(72 citation statements)

References 28 publications

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“…A Comparison of the in Vitro Uridylylation Reaction in the Presence and Absence of an RNA Template-The presence of an RNA template is an absolute requirement for PV VPg uridylylation, in contrast to rabbit hemorrhagic disease virus (8,28). Our result suggests that under the experimental conditions used, the PVA system did not require a template.…”

Section: Resultsmentioning

confidence: 72%

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 72%

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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“…The lack of a permissive cell culture for RHDV has impeded most of the molecular studies concerning the mechanisms of viral replication, although significant advances have been made in recent years using recombinant purified viral products (26,27,46,47).…”

Section: Discussionmentioning

confidence: 99%

Synthesis in Vitro of Rabbit Hemorrhagic Disease Virus Subgenomic RNA by Internal Initiation on (–)Sense Genomic RNA

Morales¹,

Bárcena²,

Ramírez³

et al. 2004

Journal of Biological Chemistry

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Rabbit hemorrhagic disease virus (RHDV), a positivestrand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3 one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (؊)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (؊)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (؉1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.Positive-strand RNA viruses are defined by the translatability of their genomic RNAs (gRNA).1 For some of them, such as picornaviruses and flaviviruses, the gRNA is the only viral mRNA in the infected cells. Nevertheless, there are many other viruses in which one or more subgenomic mRNAs (sgRNA) are also synthesized. SgRNA of positive-strand viruses are 3Ј-coterminal and identical to the gRNA for most of their length but have deletions at the 5Ј ends (with respect to gRNA) to bring their 5Ј ends close to the start codon of downstream (on genomic RNA) ORFs. In these cases, some of the viral genes are translated from the sgRNA species, thus creating the potential for independently regulating the levels of viral proteins in infected cells. SgRNA synthesis has been more studied in plant viruses than in animal viruses, probably because a greater percentage of all of the plant viruses make sgRNA and also because their smaller genomes and highly efficient replication make them more amenable to studies on RNA replication mechanisms, especially using cellfree extracts. Several basic mechanisms for generating subgenomic RNAs have been defined (1). The most widely recognized model is internal initiation on longer-than-subgenomic-length (Ϫ)strand templates in which the RNA-dependent RNA polymerase (RdRp) internally initiates (ϩ)strand sgRNA synthesis. Initiation of RNA synthesis occurs at selected sites called promoters, which have been characterized in several plant viruses (2-11) and the alphavirus (12). Most of the characterized viral RNA promoters are located at the 3Ј end of the RNA templates, and they often have stem-loop structures or consist of short single-stranded regions with un...

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“…The investigation of enzyme reactions relevant to virus replication, such as RNA synthesis initiation and elongation or the role of VPg in genome replication, which have been extensively studied in the poliovirus system using in vitro cell free systems (Cello et al, 2002;Franco et al, 2005;Molla et al, 1991) have been mostly addressed in caliciviruses using recombinant proteins in vitro (López Vázquez et al, 1998;Machín et al, 2001;Marin et al, 2000;Morales et al, 2004), in the absence of other viral and cellular components which might be crucial for the specificity and efficiency of such processes. The isolation of replication complexes from virus-infected cells could provide a valuable tool for studying, not only the macromolecular components involved in such membranous structures but as an experimental system in which various parameters and or components could be accessible to external manipulation.…”

Section: Discussionmentioning

confidence: 99%

“…Uridylylation of RaV genome-linked protein (VPg) in crude membrane fractions was analysed using a previously described protocol (Machín et al, 2001). Briefly, the reaction (20 l final volume) included 50 mM HEPES pH 7.5, 1 mM MnCl 2 , 2 Ci [␣-32 P]-UTP (800 Ci mmol −1 ), and appropriate amounts of RaV RCs (15.6 g) or exogenously added recombinant VPg (1.4 g).…”

Section: Vpg Uridylylation Assaymentioning

confidence: 99%

“…The isolation and characterization of enzymatically active RCs (Green et al, 2002) has opened new perspectives for the study of calicivirus replication in a system in which relevant protein-protein interactions could be reasonably preserved (Kaiser et al, 2006). The use of such complexes is a step forward from the extremely simplified conditions, based on purified recombinant proteins, used in most of the published studies addressing calicivirus replication López Vázquez et al, 1998;Machín et al, 2001;Wei et al, 2001) or those based on the use of heterologous hosts (Kaiser et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells

et al. 2008

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a b s t r a c tRabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B-and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

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Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation

Cited by 51 publications

References 28 publications

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Synthesis in Vitro of Rabbit Hemorrhagic Disease Virus Subgenomic RNA by Internal Initiation on (–)Sense Genomic RNA

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells

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