Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity.

Positive-strand RNA virus genomes are translated into polyproteins that are processed by viral proteases to yield functional intermediate and mature proteins. Coronaviruses (CoVs) carry genes that encode an nsp5 protease (also known as 3CLpro or Mpro) responsible for 11 maturation cleavages. The nsp5 structure contains two chymotrypsin-like domains (D1 and D2) and a unique domain (D3), and forms functional dimers. However, little is known of interactions or communication across the structure of the protease during nsp5 activity. Using reverse genetic mutagenesis of the CoV murine hepatitis virus (MHV) nsp5, we identified a new temperature-sensitive (ts) mutation in D2 of nsp5 (Ser133Ala) and confirmed a ts residue in D3 (Phe219Leu). Both D2-tsS133A and D3-tsF219L were impaired for viral replication and nsp5-mediated polyprotein processing at the nonpermissive temperature. Passage of tsS133A and tsF219L at the nonpermissive temperature resulted in emergence of multiple second-site suppressor mutations, singly and in combinations. Among the second-site mutations, a D2 His134Tyr change suppressed the ts phenotype of D2-tsS133A and D3-tsF219L, as well as the previously reported D2-tsV148A. Analysis of multiple CoV nsp5 structures, and alignment of nonredundant nsp5 primary sequences, demonstrated that ts and suppressor residues are not conserved across CoVs and are physically distant (>10 Å) from each other, from catalytic and substrate-binding residues, and from the nsp5 dimer interface. These findings demonstrate that long-distance communication pathways between multiple residues and domains of nsp5 play a significant role in nsp5 activity and viral replication, suggesting possible novel targets for non-active site inhibitors of nsp5.

Acoustic and perceptual analysis of the sibilant sound /s/ before and after orthognathic surgery.

PURPOSE: Orthognathic surgery may have a positive or negative effect on speech. Perceptual evaluation of presurgical and postsurgical articulation is difficult because speech errors, when they occur, are usually fricative distortions, which may be difficult to document reliably. In this study, acoustic analysis was used to supplement perceptual judgment of presurgical and postsurgical productions of /s/. SUBJECTS AND METHODS: The study population consisted of 9 Cantonese speakers undergoing osteotomy for Class III skeletal deformity and 9 age- and gender-matched adults with normal occlusion and speech. The speech sample consisted of 6 words with the initial sibilant sound /s/. Perceptual analysis included narrow phonetic transcription and classification of error types. Acoustic analysis included measurement of first and second spectral peaks, fricative duration, noise bandwidth, and noise-to-vowel decibel ratio. RESULTS: The results of the perceptual analysis showed a decrease in articulatory errors for the group after surgery, although 5 patients had no perceptual errors before surgery. Acoustic analysis showed significant differences between the experimental and control groups before surgery for 2 variables (spectral peak I and bandwidth). Three months after surgery there were no significant differences between the control group and the experimental group, except for bandwidth. Twelve months after surgery, there were significant differences between the 2 groups in noise bandwidth and spectral peak II. CONCLUSIONS: The results suggest a possible relapse at 1 year after surgery, based on spectral peak values. Osteotomy appears to result in a positive change in articulation for most patients, but speech outcome after osteotomy must be evaluated both 1 year and shortly after surgery.