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Category: Viruses and Viral Pathogenesis
The Arterivirus Replicase, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap06-1.gif /docserver/preview/fulltext/10.1128/9781555815790/9781555814557_Chap06-2.gifAbstract:
This chapter focuses on the arterivirus proteins that are involved in genome replication and subgenomic RNA (sgRNA) synthesis. These proteins, which are collectively referred to as “replicase/transcriptase” or—for simplicity—just “replicase,” are encoded by open reading frames 1a and 1b (ORF1a and ORF1b) of the arterivirus genome. Comparative sequence analysis of the various prototypic arterivirus genomes, and comparison with more distantly related nidoviruses, led to the identification of a number of conserved domains within the replicase. The arterivirus replicase gene covers approximately 75% of the genome. The chapter provides overviews of ORF1a- and ORF1b-encoded conserved domains. Translation of ORF1b requires a –1 ribosomal frameshift in the region just upstream of the ORF1a termination codon, resulting in the C-terminal extension of pp1a to give pp1ab. Proteolytic processing plays an important role during the arterivirus life cycle and presumably is a way to control the temporal and spatial release of replicase subunits. The proteolytic processing of the replicase polyproteins controls expression (in time and space) of the various replicase processing intermediates and end products. The chapter also talks about the properties of replicase subunits, and the arterivirus replication/transcription complex.
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Schematic representation of the replicases of the arteriviruses EAV, SHFV, LDV, and PRRSV. Sequences are aligned on the ribosomal frameshift site (RFS), and nsp numbers are shown above each sequence. The (putative) PCPα, PCPβ, PCPγ, and CP cleavage sites are indicated with open triangles, and the 3CLSP cleavage sites are indicated with black triangles. Conserved domains are indicated with black and gray boxes. ZF, putative zinc finger; C/H, cysteine/histidine-rich clusters; TM, predicted transmembrane regions; P, protease domain; Z, ZBD; N, NendoU. The diagram was based on the EAV Bucyrus strain (Swiss-Prot accession no. P19811) ( 16 ), SHFV strain LVR 42-0 (Q68772), the LDV P strain (Q83017) ( 38 ), and PRRSV strain VR-2332 (Q9WJB2) ( 35 ).
RNA sequence of the ORF1a/1b ribosomal frameshift region and the predicted frameshift-regulating RNA pseudo-knot structure. The proposed slippery sequence is indicated with a gray box; the ORF1a stop codon (bold) is underlined. The structures were adapted from or modeled based on references 16 , 18 , and 33 . The SHFV structure was modeled using sequence data from GenBank accession no. AF180391.
Proteolytic processing of arterivirus pp1ab polyproteins. Cleavages that only occur as part of the minor pathway ( 74 ) are indicated in italics. The P1 and P1’ residues of the C-terminal cleavage site of each nsp are indicated in single-letter amino acid code; for SHFV, PRRSV, and LDV, these are predicted sites based on comparative sequence analysis. Cleavage sites for various nsp1 PCP domains are unknown and therefore indicated with a question mark. The data and numbering relate to the EAV Bucyrus strain (Swiss-Prot accession no. P19811), SHFV (Q68772), the PRRSV Lelystad strain (Q04561), and the LDV P strain (Q83017).
Proteolytic processing of EAV replicase pp1a via the major and minor pathways, of which the former depends on an interaction between nsp2 and nsp3-8 ( 74 ). The proteases involved in processing are indicated with a white P in a black box. Sites cleaved by PCPβ and by CP are indicated by open triangles. Cleavages mediated by the 3CLSP nsp4 are indicated by black triangles. TM indicates predicted transmembrane domains.
Alignment of the putative nsp1 zinc finger and PCP domains of arteriviruses. Conserved residues are shown in black. Residues 75 and 50% conserved are indicated in dark and light gray, respectively. Numbers refer to the amino acid position in the pp1ab sequence. (A) Conserved Cys (or His) residues in the putative zinc finger domain that might be involved in metal ion coordination are indicated with asterisks. Active-site Cys and His residues in the PCPα (B) and PCPβ (C) domains are indicated with pound signs. The missing catalytic Cys in EAV PCPα is indicated with an “X” above the sequence. SHFV contains an additional PCPγ domain, which could be aligned with the PCPβ domains of the other arteriviruses. Alignments were generated and edited with MAFFT ( 28 ) and the GeneDoc software, using sequence data from the PRRSV Lelystad strain (Swiss-Prot accession no. Q04561) and the sequences of other arteriviruses as indicated in the legend to Fig. 1 .
Nuclear and perinuclear localization of nsp1 in EAV-infected cells. Shown is immunofluorescence staining of EAV-infected BHK-21 cells, 8 h after infection. Nuclei were stained with Hoechst 33528 (A), and nsp1 was detected using a rabbit polyclonal antiserum (B).
Multiple alignment of the arterivirus nsp2 CP domain. The Cys and His residues of the catalytic dyad are indicated with pound signs. Conserved Cys residues that are required for activity in EAV are indicated with asterisks. Numbers refer to the amino acid position in the pp1ab sequence. For further details, see the legend to Fig. 5 .
Multiple alignment of the 3CSLP (main protease) domain of arterivirus nsp4 proteins. The conserved residues of the catalytic triad are indicated with pound signs. Residues predicted to be involved in substrate recognition are indicated with asterisks. Numbers refer to the amino acid position in the pp1ab sequence. For further details, see the legend to Fig. 5 .
Structural model of EAV nsp4, the main protease. The N- and C-terminal β-barrels and the C-terminal extension are indicated above the model. Catalytic triad residues (thick black sticks) are indicated with white text in a black box. Residues involved in substrate recognition (thin black sticks) are indicated by black text. Numbers refer to the amino acid position in the nsp4 subunit. The ribbon model was prepared with Pymol (Delano Scientific) and PDB entry 1MBM.
Multiple alignment of arterivirus nsp10 domains. (A) Putative ZBD. Conserved Cys and His residues are indicated with asterisks. The Ser in the putative hinge region is boxed (hatched) (B) The NTPase/HEL domain. The NTP-binding motif is underlined, and residues that are conserved in type 1 superfamily HELs are indicated with asterisks. For further details, see the legend to Fig. 5 .
Comparison of arterivirus replicase gene features