RNA Synthesis Phenotype of the Alb <i>ts</i>22 Mutant

<p>RNA synthesis was determined (A) using 1 h pulse labels with ³H-uridine in the presence of dactinomycin, given to MHV-A59-, Alb <i>ts</i>22-, and Alb 22R-infected cells 1–6 hpi at 40 °C or 5–14 hpi at 30 °C; █, 40 °C, wt MHV-A59; ▴, 40 °C, Alb 22R; •, 40 °C, Alb <i>ts</i>22; □, 30 °C, wt MHV-A59; ▵, 30 °C, Alb 22R; ○, 30 °C, Alb <i>ts</i>22, or (B) using 30 min pulse labels with ³H-uridine in the presence of dactinomycin, given to MHV-A59-, Alb <i>ts</i>22-, and Alb 22R-infected cells after shift from the permissive to the non-permissive temperature at 13 hpi; █, wt MHV-A59; ▴, Alb 22R; •, Alb <i>ts</i>22.</p

RNA Synthesis Phenotype of MHV-A59 <i>ts</i> Mutants

<p>RNA synthesis was determined using a 1 h pulse label with ³H-uridine in the presence of dactinomycin and cycloheximide, given to wt MHV-A59 and <i>ts</i> mutant virus-infected cells at 8 hpi with or without shifting from the permissive to the non-permissive temperature. The amount of incorporated ³H-uridine at 40 °C was divided by that at 30 °C and 1.0 was subtracted. The results represent the average of five separate experiments. A value of zero means the incorporation at the two temperatures was the same.</p

Organization and Expression of the MHV-A59 Genome

<p>The structural relationships of the MHV-A59 genome and sub-genomic mRNAs are shown. The virus ORFs are depicted as lightly shaded (replicase proteins), shaded (accessory proteins), and heavily shaded (structural proteins). The ORFs are defined by the genomic sequence of MHV-A59 as published by Coley et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010039#ppat-0010039-b045" target="_blank">45</a>]. The hatched box represents the common 5′ leader sequence and the hatched circle represents the programmed (−1) frameshifting element. The translation products of the genome and sub-genomic mRNAs are depicted and the autoproteolytic processing of the ORF1a and ORF1a/ORF1b polyproteins into non-structural proteins nsp1 to nsp16 is shown. A number of confirmed and putative functional domains in the non-structural proteins are also indicated: 3CL, 3C-like cysteine proteinase; ExoN, exonuclease; HEL, superfamily 1 helicase; MT, S-adenosylmethionine-dependent 2′-<i>O</i>-methyl transferase; NeU, endoribonuclease; PL1, papain-like protease 1; PL2, papain-like protease 2; POL, RNA-dependent RNA polymerase; X, adenosine diphosphate-ribose 1′-phosphatase.</p

Biochemical Complementation Analysis of Selected MHV-A59 <i>ts</i> Mutants

<p>Cells were mock-infected or infected with MHV-A59, one of the <i>ts</i> mutants, or with a mixture of two <i>ts</i> mutants. The cells were incubated at 40 °C in medium containing dactinomycin and ³H-uridine and, at 8 hpi, ³H-uridine incorporation into trichloroacetic acid-precipitated RNA was determined. Cells were infected with: M, mock-infected; A59, MHV-A59; A6, Alb <i>ts</i>6; A16, Alb <i>ts</i>16; A22, Alb <i>ts</i>22; A17, Alb <i>ts</i>17; L6, LA <i>ts</i>6; W18, Wü <i>ts</i>18; W36, Wü <i>ts</i>36; W38, Wü <i>ts</i>38; A6xA16, Alb <i>ts</i>6 and Alb <i>ts</i>16; A6xL6, Alb <i>ts</i>6 and LA <i>ts</i>6; A6xA22, Alb <i>ts</i>6 and Alb <i>ts</i>22; A16xL6, Alb <i>ts</i>16 and LA <i>ts</i>6; A16xA22, Alb <i>ts</i>16 and Alb <i>ts</i>22; L6xA22, LA <i>ts</i>6 and Alb <i>ts</i>22; A17x A16, Alb <i>ts</i>17 and Alb <i>ts</i>16; A17xL6, Alb <i>ts</i>17 and LA <i>ts</i>6; A17xA22 or A22xA17, Alb <i>ts</i>17 and Alb <i>ts</i>22; A17xW38, Alb <i>ts</i>17 and Wü <i>ts</i>38; A17xW18, Alb <i>ts</i>17 and Wü <i>ts</i>18; A17xW36, Alb <i>ts</i>17 and Wü <i>ts</i>36; A22xW18, Alb <i>ts</i>22 and Wü <i>ts</i>18; A22xW36, Alb <i>ts</i>22 and Wü <i>ts</i>36; A22xW38, Alb <i>ts</i>22 and Wü <i>ts</i>38; W18xW36, Wü <i>ts</i>18 and Wü <i>ts</i>36; W18xW38, Wü <i>ts</i>18 and Wü <i>ts</i>38; W36xW38, Wü <i>ts</i>36 and Wü <i>ts</i>38.</p

RNA Synthesis Phenotype of the Alb <i>ts</i>16 and LA <i>ts</i>6 Mutants

<p>RNA synthesis (A) or negative-strand RNA synthesis (B) was determined using 20 or 30 min pulse labels with ³H-uridine in the presence of dactinomycin, with or without the addition of CH, after shifting the incubation temperature of MHV-A59-, Alb <i>ts</i>16-, and LA <i>ts</i>6-infected cells from 30 °C to 40 °C at 8 hpi: filled bar, 0–20 min pulse; grey bar, 20–40 min pulse; open bar, 40–60 min pulse; dark diagonal bar, 0–30 min pulse; light diagonal bar, 30–60 min pulse.</p

Genotypic Analysis of Selected MHV-A59 <i>ts</i> Mutants

<div><p>(A) The positions of mutations responsible for the <i>ts</i> phenotype of selected MHV-A59 mutants are illustrated in relation to the non-structural proteins (nsp1–16) produced by proteolytic processing of the ORF1a/ORF1b polyprotein, pp1ab. Nucleotide changes are numbered according to the sequence of the infectious cDNA clone of MHV-A59.</p><p>(B) The amino acid substitutions responsible for the mutant and revertant phenotypes are listed together with the mutated protein and the cistron to which each mutant has been assigned. The amino acids are numbered from the amino-terminus to the carboxyl-terminus of each of the non-structural proteins.</p></div

A Model to Describe the Pathway for Viral RNA Synthesis in MHV-A59-Infected Cells

<p>Shows a working model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. The model proposes that the replicase-transcriptase complex forms initially and creates a negative-strand template. It is then converted to utilize the negative strand as a template for positive-strand synthesis and, finally, the complex is inactivated by the degradation of negative-strand templates. It is also proposed that proteolytic processing of the replicase polyproteins plays a role in regulation of this pathway. Also depicted are the putative defects of specific MHV-A59 <i>ts</i> mutants. It remains to be shown whether or not the group IV and VI mutants (Wü <i>ts</i>38, Alb <i>ts</i>17, Wü <i>ts</i>18, and Wü <i>ts</i>36) are defective in negative-strand RNA synthesis at the non-permissive temperature.</p