Relationship between viral mRNA accumulation profiles and infectious virus yield.

<p>The data obtained on the accumulation of genome and sg mRNAs were used for a quantitative assessment of nsp1 mutant phenotypes in terms of (A) changes in mRNA accumulation compared to wt and (B) the extent to which the balance between viral transcripts was disturbed (B). (A) For each mutant and pseudorevertant, a value for the accumulation of each of its 7 mRNAs at 11 h post-transfection had been assigned as compared to that of the wt control (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g007" target="_blank">7B and 7D</a>). The mean of these seven values (“mean relative mRNA accumulation”) was plotted against the corresponding progeny virus titer in culture supernatants at 24 h post-transfection (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g006" target="_blank">Fig. 6B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-t002" target="_blank">table 2</a>). Wild-type is depicted in orange. The engineered nsp1 mutants and nsp1 pseudorevertants are shown in blue and purple, respectively. The three pseudorevertants of mutant A4, displaying very similar phenotypes, are indicated as A4+PSR. (B) For each mRNA species, the deviation of its relative accumulation from the mean of the complete nested set of mRNAs (see panel A) was calculated. From these seven values, the mean (absolute) deviation was calculated for each mutant and plotted against virus titers as in (A). For wt, the mean deviation is 0. Engineered nsp1 mutants and their pseudorevertants are indicated as in (A). The data fit a negative exponential regression calculated using Microsoft Excel and depicted as a gray line (y = 6×10⁶+7e^−8.4306x, R² = 0.95). The inset shows an expanded view of the upper left quadrant of the graph (shaded in gray).</p

Analysis of viral protein accumulation and virus production by selected nsp1 mutants.

<p>(A) Western blot analysis of EAV-specific protein accumulation. Cells transfected with wt or mutant viral genomes were harvested 11 h after transfection and equal amounts of total protein were analyzed with EAV-specific sera detecting nsp3, M and N, which are translated from RNAs 1, 6 and 7, respectively. The relative levels of each mRNA template (derived from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>) are indicated below the gels. Beta-actin was used as a loading control. (B) Plaque phenotype and virus titers of the ZCH, A1 and A4 mutants in comparison with wt. Plaque assays were performed on BHK-21 using cell culture supernatants harvested 24 h after transfection. Virus titers represent an average of three independent experiments.</p

Minus-strand RNA accumulation is also modulated by mutations in nsp1.

<p>(A–D) Analysis and quantification of EAV minus-strand accumulation by a two-cycle RNase protection assay. (A) Schematic representation of the nested set of viral minus-strand RNA [(−)RNA] species produced in EAV-infected cells. The anti-leader sequence is depicted in light green. The in vitro-transcribed plus-strand probes used for detection of (−)RNA1 (pRNA1), (−)RNA6 (pRNA6) and (−) RNA7 (pRNA7) are shown. pRNA6 and pRNA7 target the leader-body junction sequences of (−)RNA6 and (−)RNA7, respectively. Note that hybridization with pRNA1 results in the protection of a single fragment, while the probes for (−)RNAs 6 and 7 each protect three fragments – one derived from the full-length sg minus strand, and two fragments derived in part from partial hybridization of these probes to larger viral (−)RNAs in which the target sequences are noncontiguous (exemplified for pRNA6). For simplicity, non-EAV sequences present near the termini of the three probes were omitted from the scheme. (B) Viral (−)RNA accumulation was analyzed at 11 h post-transfection for the ZCH, A1 and A4 mutants, and a wt control. Protected fragments were resolved on denaturing 5% polyacrylamide/8M urea gels and visualized by phosphorimaging. The constructs analyzed are labeled above the lanes (M, mock-transfected cells; (−), no-RNase control that shows a band corresponding to 0.2 fmol of the full-length probe). Sizes (nt) of RNA markers have been indicated on the left. The single 327-nt protected fragment resulting from hybridization with the positive-sense probe for RNA1(−) is indicated. The probes for subgenome-length minus strands protected fragments derived from the full-length (−)RNA6 and (−)RNA7 (327 nt and 319 nt, respectively; denoted with LB), as well as from the (−)RNA6 and (−)RNA7 body sequences (188 nt and 180 nt, respectively; denoted with B) and the anti-leader sequence (139 nt; denoted with L). The presence of two bands in the size range of the anti-leader fragment has been described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat.1000772-denBoon3" target="_blank">[55]</a>. (C) The relative levels of minus-strand accumulation were quantified by phosphorimaging. For (−)RNAs 6 and 7, only the bands resulting from protection of full-length sg minus strands (denoted with LB in panel [B]) were quantified. The values correspond to the means from three independent transfections that were normalized to the level of accumulation of each minus-strand RNA in the wt control, which was set at 1. Intracellular RNA from the same transfection samples for which plus-strand accumulation was quantified (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a>) was used. Genomic minus-strand RNA levels are represented as dark blue bars. Error bars denote standard deviation. (D) The ratio of plus-strand to minus-strand accumulation for RNAs 1, 6 and 7 was calculated using the mean relative values obtained in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g004" target="_blank">Fig. 4B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g005" target="_blank">Fig. 5C</a>.</p

Domain organization of EAV nsp1.

<p>The partial sequence alignment shows key regions in the three subdomains previously identified in the arterivirus nsp1 region. GenBank accession numbers for the full-length arterivirus genomes used for the alignment are as follows: EAV, NC_002532; simian hemorrhagic fever virus (SHFV), NC_003092; lactate dehydrogenase-elevating virus (LDV-P and LDV-C), NC_001639 and NC_002534; PRRSV-LV, M96262.2; PRRSV-VR, AY150564. Zinc-coordinating residues are indicated in bold font; the active-site Cys and His of PCPα and PCPβ are indicated with triangles (note the loss of the active-site Cys in EAV PCPα). The positions of amino acid clusters mutated in this study are indicated with arrows. All substitutions were with Ala, with the exception of the ZCH construct, in which Cys-25 and His-27 were swapped. The positions of mutations found in pseudorevertants are indicated with open circles.</p

Organization and expression of the polycistronic EAV +RNA genome.

<p>(A) Top: EAV genome organization, showing the 5′-proximal replicase open reading frames (ORFs), as well as the downstream ORFs encoding the viral structural proteins envelope (E), membrane (M), nucleocapsid (N), and glycoproteins (GP) 2–5 and the 3′ poly(A) tail (A_n). Bottom: overview of the pp1a and pp1ab replicase polyproteins that result from genome translation, which requires an ORF1a/1b ribosomal frameshift (RFS) to produce pp1ab. Arrowheads represent sites cleaved by the three virus-encoded proteases (open for autoproteolytically processed ones, closed for sites processed by the main proteinase in nsp4). The resulting nonstructural proteins (nsp) are numbered. The key viral enzymatic domains such as the nsp1 papain-like cysteine proteinase β (PCP), nsp2 cysteine proteinase (CP), nsp4 serine proteinase (SP), nsp9 viral RNA-dependent RNA polymerase (RdRp), nsp10 helicase (Hel), and nsp11 endoribonuclease (Ne) are indicated. (B) Overview of viral mRNA species produced in EAV-infected cells. The ORFs expressed from the respective mRNAs are shown in gray, and the 5′ leader sequence is depicted in dark red. The orange boxes indicate the positions of transcription-regulating sequences (TRS). The gel hybridization image on the right is representative of the wild-type accumulation levels of the seven EAV mRNAs at the time point used for analysis in the study (see text for details). The amount of each mRNA, determined by quantitative phosphorimager analysis, is indicated as percentage of the total amount of viral mRNA. (C) Model for EAV replication and transcription. Continuous minus-strand RNA synthesis yields a genome-length minus strand template for genome replication, a process for which nsp1 is dispensable. Discontinuous minus-strand RNA synthesis results in a nested set of subgenome-length minus strands that serve as templates for sg mRNA synthesis (see text for details). Nsp1 is crucial for this process, which is also guided by a base pairing interaction between the TRS complement [(−)TRS] at the 3′ end of the nascent minus-strand and the genomic leader TRS, present in a RNA hairpin structure (LTH).</p

Overview of EAV nsp1 pseudorevertants described in this study.

a<p>Virus clones were isolated from plaque assays of culture supernatants harvested between 20 h and 24 h post transfection. The number of clones containing each mutation is indicated in brackets.</p>b<p>Numbers indicate the start coordinate of the codon in the EAV genome.</p>c<p>Pseudorevertants were reconstructed in the wild-type EAV background, and infectious progeny titers were determined by plaque assays of culture supernatants harvested at 24 h post-transfection. The average titers of three independent experiments are shown.</p>d<p>NA, not applicable.</p>e<p>A silent mutation changing the original alanine codon from GCG to GCC was introduced as a marker mutation upon construction of the ZCH mutant.</p

Relative specific infectivities of virus particles from nsp1 mutants with imbalanced mRNA profiles.

a<p>Virus titers were determined by plaque assays of culture supernatants harvested at 24 h post-transfection and normalized to the pfu/ml value of the wt control, which was set at 1.</p>b<p>EAV genomic RNA levels were quantified in virion preparations obtained from culture supernatants harvested at 24 h post-transfection by reverse transcription and quantitative PCR. Values for mutant virions were obtained by comparing their threshold cycle (Ct) against the qPCR standard curve, and were normalized relative to the genomic RNA level in the wt control, which was set at 1.</p>c<p>Relative specific infectivity values were calculated by dividing the relative infectivity (mutant∶wt pfu ratio) by the relative genomic RNA content for each construct.</p>d<p>The data shown are derived from two independent experiments.</p

Overview of the genotype and first-cycle phenotype of EAV nsp1 mutants described in this study.

a<p>Nucleotide substitutions are indicated in bold italics; numbers indicate the start and end coordinates of the mutated codons in the EAV genome.</p>b<p>Transfected cells were analyzed by IFA and gel hybridization analysis at 11 h post-transfection. Wild-type levels of replication and transcription (based on accumulation levels of genomic and sg mRNAs, respectively) are indicated with +++. A 2- to 4-fold increase in genomic RNA levels as compared to wild-type is denoted by ++++, while an increase of >4-fold is shown as +++++. Likewise, a >2-fold increase or decrease in accumulation levels of at least two sg mRNA species is shown as ++++ and ++, respectively (see text for details).</p>c<p>NA, not applicable.</p

Multiple mutations in nsp1 exert species-specific effects on viral mRNA accumulation.

<p>(A, B) Gel hybridization analysis and quantification of EAV-specific mRNA accumulation in cells transfected with the ZCH, A1, A4 mutant or a wt control. (A) Viral mRNA accumulation was analyzed at 11 h post-transfection by gel hybridization as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000772#ppat-1000772-g003" target="_blank">Fig. 3</a>. (B) The accumulation levels of each viral mRNA in the nsp1 mutants were quantified by phosphorimaging in the linear range of exposure and normalized to the level of accumulation of each corresponding viral mRNA in the wt control, which was set at 1. Genomic RNA levels are represented as blue bars. The relative values correspond to the means from three independent transfections and error bars denote standard deviation.</p

Electron micrographs of the P10 fractions of SARS-CoV-infected cells.

<p>Immunogold labeling of the P10 fraction of SARS-CoV-infected (A, B) or uninfected (C) Vero-E6 cells using rabbit antisera recognizing nsp3 (A, C) or nsp4 (B) was followed by negative staining. The white arrow indicates a part of the specimen clearly showing double membranes. Scale bar: 500 nm.</p