Isolation and Characterization of an Arterivirus Defective Interfering RNA Genome

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Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-24293Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-24293Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of "hinge spacer," which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.Equine arteritis virus (EAV) (17) is the prototype of the Arteriviridae, a recently established family of positive-stranded, enveloped RNA viruses (for reviews see references 47 and 53). The other three members of the Arteriviridae are Lactate dehydrogenase-elevating virus (LDV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Simian hemorrhagic fever virus (SHFV). Based on their presumed common ancestry (7), the arteriviruses have been united with the coronaviruses in the order of the Nidovirales. Important common properties of nidoviruses (Fig. 1) are the presence of a unique array of conserved domains in the viral replicase, the use of ribosomal frameshifting and proteolytic processing to regulate replicase gene expression, and the use of discontinuous transcription to generate a nested set of subgenomic (sg) mRNAs for structural protein expression (for reviews see references 15, 35, and 53).The EAV nonstructural proteins are generated by proteolytic processing of two large replica...

Section: Discussionmentioning

confidence: 92%

The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Dinten

Tol

Gorbalenya

et al. 2000

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131

“…For EAV, the 353 3Ј-most nt are required for replication and/or transcription (19). Moreover, defective interference studies indicated that ORF7, which is partly contained within this sequence, contains elements involved in these processes (18). These observations indicate that a kissing interaction between the coding and noncoding sequences might occur in this 353-nt domain.…”

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confidence: 99%

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

Verheije

Olsthoorn

Kroese

et al. 2002

We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3 noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.Positive-strand RNA viruses replicate in infected cells by a process that is mediated by RNA-dependent RNA polymerase (RdRp). In this process, genomic RNA serves as a template for the production of negative-strand antigenomic RNA, which is used in turn as a template for the synthesis of new plus strands. The process of replication requires the recruitment of the RdRp to specific sequences or structures within the templates, also known as cis-acting replication elements. These elements are usually located in the noncoding regions at the termini of the viral RNA, where RdRp complexes initiate the synthesis of plus and minus strands (2). Only a few examples are known of cis-acting replication elements that reside within a coding region (7,10,11,20).Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that belongs to the Arteriviridae family (reviewed in reference 24), together with Equine arteritis virus (EAV), Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (SHFV) (17). On the basis of their similar genomic organization and replication strategy, the arteriviruses have been grouped into the order of Nidovirales together with the coronaviruses and the toroviruses (3).The genome of PRRSV is 15.1 kb (17), of which the 5Ј two-thirds is translated into two large polyproteins. These are subsequently cleaved by virus-encoded proteases to yield at least 12 nonstructural proteins, including the viral RdRp (reviewed in reference 24). In addition, a set of subgenomic mRNAs, collectively expressing the viral structural proteins, are produced through a process of discontinuous mRNA transcription (5,8,14,16). This process has not been unraveled completely and may occur during plus-or minus-strand synthesis (reviewed in references 9, 23, and 26). Besides the coding regions, the PRRSV genome contains a 5Ј untranslated region (5ЈUTR) of 221 nucleotides (nt) (24), which carries a cap at its 5Ј end (15, 21), and a 3ЈUTR of 114 nt to which the poly(A) tail is attached (17).Little is known about the requirements for arterivi...

“…We have previously reported the construction of pEDI, a full-length cDNA copy of the EAV DI-b genome from which replication-competent EDI RNA can be transcribed in vitro ( Fig. 1) (43). As described in the introduction, the in-frame fusion of the three EAV genome segments that constitute the EDI sequence has resulted in the presence of a truncated replicase ORF, EDI-ORF, that starts at the natural ORF1a translation initiation codon (nt 225) and ends at the ORF1b termination codon (nt 2576 of the EDI sequence; nt 9749 of the EAV genome).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we have described the generation of DI-b, a natural EAV DI RNA of 5.6 kb, and we have reported the construction of pEDI, a full-length cDNA copy of EAV DI-b RNA from which replication-competent RNA can be transcribed in vitro (43). We have used this EDI RNA for deletion mutagenesis, and in this way the sequences that are likely to be required for efficient replication of EAV DI RNAs were reduced to at most 589 and 1,068 nt of the genomic 5Ј-and 3Ј-terminal regions, respectively, and a segment of at most 583 nt from the 3Ј part of replicase ORF1b (43).…”

mentioning

confidence: 99%

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Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs

Molenkamp

Greve

Spaan

et al. 2000

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Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3-and 5-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5-proximal region was found to be approximately 100-fold lower than that in the 3-proximal part of the genome.Equine arteritis virus (EAV) is the prototype member of the family Arteriviridae (52). Based on similarities in genome organization, protein expression strategies, and the presumed common ancestry of their replicase genes (12), the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales (7,15).EAV is a spherical, enveloped RNA virus (for a review, see reference 52) and contains a positive-strand genome of approximately 12.7 kb (12). The virion envelope is derived from intracellular host cell membranes and contains five or six structural proteins (14,52,53). The envelope surrounds an isometric nucleocapsid, which is composed of the genomic RNA and multiple copies of the nucleocapsid (N) protein.The EAV replicase is translated in the form of two large polyproteins, the open reading frame 1a (ORF1a) and ORF1ab proteins. The C-terminal part of the latter is produced by an ORF1a/1b ribosomal frameshift (12). Extensive studies on the proteolytic processing of the ORF1a and ORF1ab polyproteins have resulted in an apparently complete processing scheme (54,62,63,66), which comprises the production of 12 end products (nsp1 to nsp12) and a large number of processing intermediates.As in coronaviruses, the EAV structural proteins are translated from a 3Ј-coterminal nested set subgenomic mRNAs (sg mRNAs), which also share a common 5Ј-leader sequence that is derived from the 5Ј end of the genome (12, 13, 52...