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Short Communication |
and nsp1β papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus RNA synthesis
1 Wageningen UR, Animal Sciences Group, Division of Infectious Diseases, PO Box 65, 8200 AB Lelystad, The Netherlands
2 Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
Correspondence
Lisette A. H. M. Cornelissen
lisette.cornelissen{at}wur.nl
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ABSTRACT |
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and nsp1β, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCP and PCPβ, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCP and PCPβ were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCP activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPβ activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1β/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1/nsp1β and nsp1β/nsp2 junctions.
Present address: Genmab, PO Box 85199, 3508 AD Utrecht, The Netherlands. 
Present address: Wageningen UR, Central Institute for Animal Disease Control Lelystad, CIDC-Lelystad, PO Box 2004, 8203 AA Lelystad, The Netherlands.
Present address: Amsterdam Molecular Therapeutics, PO Box 22506, 1100 DA Amsterdam, The Netherlands.
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).
Following virus entry and release of the genome into the cytoplasm, the nidovirus life cycle starts with the expression of the large replicase gene that consists of open reading frame (ORF) 1a and ORF1b. Genome translation yields two multidomain replicase polyproteins, named pp1a and pp1ab, with the latter being a C-terminally extended version of the former due to a ribosomal frameshift mechanism (Fig. 1). Polyproteins pp1a and pp1ab are co- and post-translationally processed by the viral ‘main’ proteinase and one to three ‘accessory’ autoproteinases (reviewed by Ziebuhr et al., 2000). The resulting mature nsps direct viral RNA synthesis, presumably after forming a RTC that is associated with endoplasmic reticulum-derived paired membranes and double membrane vesicles (Gosert et al., 2002; Pedersen et al., 1999). The sg mRNAs of arteri- and coronaviruses consist of a 5'-terminal common ‘leader’ sequence, derived from the 5' nontranslated region (NTR) of RNA1, that is fused to different 3'-proximal regions of the genome, the ‘mRNA bodies’. The 5'-proximal cistron of each sg mRNA is translated to produce the viral structural proteins. The subgenome-length templates for sg mRNA synthesis are thought to be generated by discontinuous minus-strand RNA synthesis during which sequences that are non-contiguous in the genome are joined (reviewed by Pasternak et al., 2006). This leader-to-body fusion event is mediated by transcription-regulating sequences that are located at the 3'-end of the leader and upstream of all genes encoding the structural proteins. Additional factors such as viral nsps, presumably host proteins, and higher order RNA structures that direct leader-to-body fusion are thought to be involved (van den Born et al., 2004).
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, nsp1β and nsp2 (van Aken et al., 2006; Ziebuhr et al., 2000). Papain-like proteinase (PCP) directs the release of nsp1, whereas the liberation of nsp1β depends on the activities of both PCP and a second proteinase, PCPβ (Fig. 1
). A third cysteine proteinase, residing in the N-terminal domain of nsp2, is responsible for the cleavage of the nsp2/nsp3 site (den Boon et al., 1995; Snijder et al., 1995). Viral papain-like proteinases are characterized by a catalytic dyad that consists of a nucleophilic cysteine residue and a downstream histidine. Based on comparative sequence analysis and in vitro activity assays, Cys-276 and His-345 were proposed to form the catalytic dyad of PRRSV PCPβ. Cys-76 and His-146 form the putative catalytic dyad of PRRSV PCP. Mutagenesis of two other PCP His residues (His-115 and His-157) partially inhibited proteolytic activity in vitro. EAV PCP has lost its proteolytic activity since the equivalent of PRRSV Cys-76 is no longer present (den Boon et al., 1995). Comparative sequence analysis among arteriviruses identified a putative zinc finger domain near the N terminus of EAV nsp1 (Tijms et al., 2001, 2007) and a C-terminal PCP domain (PCPβ) (den Boon et al., 1995; Snijder et al., 1994).
To investigate the importance of the PCP and PCPβ autoproteinase domains in the PRRSV life cycle, their putative active-site residues were probed by site-directed mutagenesis in the context of a PRRSV reverse genetics system (Meulenberg et al., 1998). The predicted catalytic residues of both PCP domains were substituted by residues that completely inactivated proteolytic activity in vitro. Also PCP residues His-115 and His-157 were probed by substituting them with residues that still allowed partial PCP activity in vitro (den Boon et al., 1995). These mutations (Table 1) were cloned into pABV437, an infectious cDNA clone of a European PRRSV strain (Meulenberg et al., 1998). In vitro-transcribed full-length PRRSV RNA was electroporated into baby hamster kidney-21 (BHK-21) cells, which support replication and yield progeny virus, but are not permissive to infection with PRRSV. Consequently, virus harvested from transfected BHK-21 cells needs to be amplified in permissive cells, like porcine alveolar macrophages (PAMs).
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mutants (Table 1; Fig. 2a), immunostaining of transfected cells revealed abundant expression of replicase subunit nsp3, which was used as a marker for genome replication. At 24 h post-transfection (h p.t.), the number of positive BHK-21 cells and their staining pattern were comparable to that of the wild-type control, indicating that RNA1 replication was not dramatically affected by the PCP mutations. As a negative control, we used a control cDNA clone that lacked an essential signal for RNA synthesis in its 3'-proximal domain (data not shown; the construct lacks nt 11786–14581). The fact that no positive cells were observed following transfection of this replication-incompetent RNA indicated that translation of the input RNA in itself did not yield detectable levels of nsp3 expression, since the replicase gene of the deletion mutant was intact and available for translation. Thus, the detection of nsp3 by immunostaining could indeed be used as a read-out for successful RNA1 replication. In addition, intracellular RNA isolated from BHK-21 cells that had been transfected with a selection of mutants was used for RT-PCR analysis. Antisense oligonucleotide LV388 (nt 15 067–15 098; 5'-AATTTCGGTCACATGGTTCCTGCCTGATTAAG-3') or LV385 (nt 224–250; 5'-CACATGCACCGGGAGAACGTCCCAGAC-3') was used in the reverse transcription reaction, followed by a PCR in which the leader-specific (sense) oligonucleotide LV383 (nt 1–29; 5'-ATGATGTGTAGGGTATTCCCCCTACATAC-3') was added to produce either a 252 bp fragment from RNA1 or a 733 bp fragment from sg mRNA7 (Fig. 2b). For all mutants, RNA1 amounts were detected similar to those of the wild-type control (Fig. 2c), confirming that replication was indeed not affected.
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mutants, the synthesis of the nucleocapsid protein (N), which is expressed from the smallest and most abundant sg mRNA (sg mRNA7), was analysed by immunostaining at 24 h p.t. (Table 1; Fig. 2a). Only a small number of BHK-21 cells transfected with any of the PCP mutants were positive for N, and in these cells the intensity of the N-staining was comparable to that in cells transfected with the wild-type control. When comparing the signal for nsp3 and N in double staining experiments, only a few of the nsp3-positive cells were also positive for N. Mutants His-115
Tyr and His-157Asn produced N-positive cells in all experiments, whereas transfections with the Cys-76Ser, His-146Tyr, His-157Asp and His-157Ile mutants resulted in the occasional appearance of a few double-positive cells, which was likely due to reversion (see below). Intracellular RNA from cells transfected with selected mutants was analysed for the synthesis of sg mRNA7 by means of RT-PCR (Fig. 2b), which confirmed that sg mRNA synthesis was severely affected in these mutants, since no signal for mRNA7 could be detected (Fig. 2c), not even when an increased number of cycles was used for the PCR. The same was true for samples that were harvested after an additional 24 h incubation (data not shown).
To test for the production of progeny virus, the supernatants of transfected BHK-21 cells were transferred to PAMs (p1). Some N-positive cells were present at 24 h post-inoculation in PAM cultures infected with the harvest from cells transfected with mutants Cys-76Ser and His-146Tyr (Table 1). The nsp1 region containing the original mutation was amplified from genomic RNA and codons 76 and 146 were found to differ by one nucleotide from the sequence in the original mutant cDNA clones. In both cases, the codon specifying the wild-type amino acid at that position had been restored (Table 1), strongly supporting the importance of Cys-76 and His-146 for PRRSV viability. Mutant His-157Asn produced N-positive cells in all passaging experiments, whereas the other substitution mutants occasionally yielded positive cells, but to a much lesser extent than the wild-type control and His-157Asn (Table 1). To assess the genetic stability of substitution mutants giving partial cleavage of the nsp1/nsp1β junction (His-115Tyr, His-157Asp, His-157Asn and His-157Ile), the p1 supernatants were used to passage these mutant viruses four more times. Subsequently, the genome sequence of the p5 viruses at the site of mutation was determined. Revertants of the His-157Asn mutant contained the wild-type codon (true revertant), whereas mutants His-115Tyr, His-157Asp and His-157Ile yielded pseudo-revertants. Mutant His-115Tyr changed to Asn, His-157Asp to Tyr, and His-157Ile reverted to Phe (Table 1). Titres of the p5 viruses were similar to that of the wild-type control (Table 1). As an alternative approach to probe the importance of His-115 and His-157, the corresponding codons were deleted (mutants 
His-115 and His-157). RNA derived from these two mutant clones did replicate in BHK-21 cells, but did not produce detectable levels of sg mRNA7, as established by immunostaining and RT-PCR analysis (Table 1; Fig. 2c). Subsequently, the mutations recovered from the p5 populations were investigated for their effect on cleavage of the nsp1/nsp1β junction. To this end, mutations specifying His-115Asn and His-157Phe, as well as the deletions of both codons, were introduced individually into expression vector pIP627 and used to translate the PRRSV nsp1 region in vitro (rabbit reticulocyte lysate) in the presence of [35S]methionine (den Boon et al., 1995). The polyprotein consisted of nsp1 (20 kDa), nsp1β (27 kDa) and the N-terminal part of nsp2 (30 kDa). Translation products were resolved in a 15 % gel by SDS-PAGE and visualized by autoradiography (Fig. 2d). The His-157Tyr mutant was not included, as den Boon et al. (1995) had already shown that this mutation did not affect the nsp1/nsp1β cleavage. Whereas mutant His-115Tyr produced only trace amounts of cleavage products, the His-115Asn mutant retained wild-type proteolytic activity. In addition, mutant His-157Ile showed no cleavage of the nsp1/nsp1β junction, whereas pseudo-revertant His-157Phe had wild-type activity. In accordance with the results obtained in the in vivo analysis, deletion of His-115 or His-157 completely abolished processing of the nsp1/nsp1β junction.
As for PCP, substitutions targeting either residue of the presumed PCPβ catalytic dyad were tested as well. Den Boon et al. (1995) had already described the complete inhibition of PCPβ proteolytic activity in vitro due to the substitutions Cys-276Leu, Cys-276Ser and His-345Tyr. In vivo, none of these mutants showed any sign of replication, as evidenced by (i) the lack of signal in immunostaining using the anti-nsp3 serum, (ii) the absence of RT-PCR signal for genome RNA (Table 1; Fig. 2c), and (iii) the absence of infectious progeny in the medium harvested from transfected BHK-21 cells. The small amounts of 252 bp RNA1-derived RT-PCR product seen in the upper panel of Fig. 2(c) (RNA1) should be attributed to amplification from remaining transfected input RNA and not to newly synthesized RNA1, as evidenced by RT-PCR analysis of RNA isolated from the supernatants of BHK-21 cells transfected with RNase-free DNase I-treated RNA transcripts using the same wild-type and negative control as well as a transcript lacking the 3' NTR (data not shown).
The data presented above indicate that PCP activity is required for PRRSV sg mRNA synthesis, most likely because the function of nsp1, which contains the putative zinc finger domain that was implicated in EAV sg RNA synthesis, depends on the release of this subunit from the replicase polyproteins. However, genome replication can proceed normally when PCP is completely inhibited, a phenotype also previously observed for EAV mutants lacking nsp1, leading to the conclusion that the protein is critical for sg mRNA synthesis, but completely dispensable for genome replication (Tijms et al., 2001, 2007). Theoretically, the effect of PCP inactivation could also be explained by a negative effect on PCPβ activity towards the nsp1β/nsp2 site, which was here shown to be absolutely required for genome replication (Table 1). However, in previous studies, den Boon et al. (1995) did not observe any effect of PCP mutations on processing of the nsp1β/nsp2 junction in vitro. An effect of the PCP mutations on the functionality of the nsp1 zinc finger domain could also explain the transcription-negative phenotype, but this is deemed less likely since the corresponding domain in EAV nsp1 that does not contain an active PCP proteinase is functional.
The PCPβ knockout mutants tested in this study, which were completely blocked in processing of the nsp1β/nsp2 junction in vitro (den Boon et al., 1995), showed no sign of RNA synthesis at all. These results, together with data obtained for EAV (Tijms et al., 2007) showing that the nsp1/nsp2 cleavage is required for virus viability, suggest that the liberation of the nsp2 N-terminus is essential for arterivirus replication. EAV nsp2 has been shown to be involved in at least three processes; (i) cleavage of the nsp2/nsp3 junction in cis by the cysteine proteinase in its N-terminal domain (Snijder et al., 1995), (ii) acting as cofactor for the nsp4 proteinase (Wassenaar et al., 1997), and (iii) formation of the double-membrane structures with which the RTC appears to be associated (Snijder et al., 2001). It remains to be established whether one or more of these functions can be extrapolated to PRRSV and how inactivation of PCPβ may affect these functions. Apart from containing the proteinase performing the important nsp1β/nsp2 cleavage, nsp1β likely has an additional role in the PRRSV life cycle, but no indications about the nature of this function have been obtained so far.
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ACKNOWLEDGEMENTS |
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Received 20 June 2007;
accepted 8 October 2007.
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