Arterivirus subgenomic mRNA synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease

Abstract

Many groups of plus-stranded RNA viruses produce additional, subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plus-stranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5'- and 3'-coterminal subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3'- and 5'-proximal domains of the genomic template. Nidovirus genome amplification ("replication") and subgenomic mRNA synthesis ("transcription") are driven by 13 to 16 nonstructural proteins (nsp's), generated by autocatalytic processing of two large "replicase" polyproteins. Previously, using a replicon system, the N-terminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis.

FIG. 1.

Schematic diagram of the genome organization and expression of EAV. The regions encoding the replicase polyproteins (pp1a and pp1ab) and structural proteins are indicated on the genome. The replication/transcription complex, presumably containing the full-length and subgenome-length minus-strand templates for replication and transcription, is depicted below the genome. sg mRNAs, with the black boxes representing their common 5′ leader sequence, are shown in the bottom part of the scheme.

FIG. 2.

(A) Processing scheme of the EAV replicase, depicted in the form of the pp1ab polyprotein. The three viral protease domains (the nsp1 PCPβ [β], the nsp2 cysteine proteinase [CP], and the nsp4 serine proteinase [S]) and their cleavage sites are indicated. Other domain abbreviations are as follows: ZF, nsp1 zinc finger domain; α, nsp1 PCPα domain; H, hydrophobic domains; RdRp, RNA-dependent RNA polymerase (nsp9); M, metal-binding domain (nsp10); Hel, helicase (nsp10); N, nidovirus-specific endoribonuclease NendoU (nsp11). (B) Schematic representation of the putative nsp1 ZF domain, showing the four residues that are proposed to coordinate zinc in gray. (C) Comparison of key sequences of the three domains identified in the arterivirus nsp1 region, ZF, PCPα, and PCPβ. The putative zinc-coordinating residues of the ZF domain and the active-site Cys and His residues of the two PCP domains are indicated. Note that EAV PCPα is no longer active due to the loss of its active-site Cys (9). Residues targeted by mutagenesis in this study are indicated with an asterisk.

FIG. 3.

(A) Northern blot analysis of genome (RNA1) and sg mRNA (RNAs 2 to 7) synthesis in BHK-21 cells transfected with EAV nsp1 ZF mutants (14 hpt) and the RNAko mutant designed to probe the role of the RNA structure of the ZF-coding region (see text). RNA was isolated at 14 hpt, and hybridization was carried out using a ³²P-labeled oligonucleotide probe complementary to the 3′ end of the genome and thus recognizing all viral mRNA species. Note the upregulation of genome synthesis in transcription-negative mutants. (B) PhosphorImager analysis of the ratio of sg mRNA to genome accumulation, based on the gel depicted in panel A. For the transcription-positive mutants, the amount of genome was put at 1 and the relative amounts measured for mRNA7, mRNA6, and mRNAs 2 to 5 are shown. With the exception of the His-27→Arg mutant, all mutants were concluded to maintain fairly normal transcription levels. wt, wild type.

FIG. 4.

Plaque phenotype of selected mutants from this study. Virus was harvested from transfected BHK-21 cells at 18 hpt. Plaque assays were also performed on BHK-21 cells and fixed after 3 days. The small-plaque phenotype of the Cys-25→His and His-27→Cys mutants is illustrated, with the latter showing sign of probable rapid reversion (plaques marked R). The fourth photograph illustrates the wild-type plaque phenotype of the RNAko mutant.

FIG. 5.

In vitro assay for PCPβ activity in nsp1 ZF mutants. Expression construct pEAVΔH (see Materials and Methods) was used to in vitro translate an nsp1-containing fusion protein in the presence of [³⁵S]methionine. Reaction products were analyzed by SDS-PAGE and autoradiography. Cleavage of the 52-kDa precursor (P) yields nsp1 and a 22-kDa C-terminal fragment (C). All nsp1 ZF mutants tested showed wild-type nsp1 PCPβ activity, thus ruling out that a defect in nsp1/2 cleavage was the basis for their phenotype.

FIG. 6.

(A) Sequence alignment of the nsp1 ZF-coding domain for a selection of EAV isolates (for details, see reference 64). The codons for the four key residues of the nsp1 ZF are boxed. (B) Structure of a predicted RNA hairpin that can be formed by the sequence specifying nsp1 ZF residues 22 to 28, thus including the highlighted codons for Cys-25 and His-27. Natural sequence variations (see panel A) are indicated in gray. Residues targeted for mutagenesis in the RNAko mutant (see text) are indicated with asterisks.