The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.

Avian infectious bronchitis virus (IBV) is a member of theCoronaviridae (order Nidovirales) (9), members of which are enveloped viruses with single-stranded, positive-sense RNA genomes that are 5Ј capped and 3Ј polyadenylated (30, 63). The 5Ј two-thirds of the coronavirus genome encodes the replicase gene producing two polyproteins, Rep1a and Rep1ab, the latter resulting from a Ϫ1 frameshift (7). The remaining proteins, which include the nucleoprotein (N), are expressed from a nested set of subgenomic mRNAs (sgRNAs) that are produced via a discontinuous transcription mechanism (6, 30). Each of these sgRNAs has a short nontranslated leader sequence (64 nucleotides for IBV) derived from the 5Ј end of the genome. Present within the leader sequence is a consensus sequence, which we have termed the transcription-associated sequence (TAS) (24). The TAS contains a conserved core motif, which in the case of IBV is CUUAACAA, which is also located in the genome, proximal to the start site for each sgRNA. For different coronaviruses, the core sequence varies and can be present more than once per TAS.N protein, the virus RNA binding protein, is one of the most abundant viral proteins in an infected cell (31). Several functions have been postulated for the coronavirus N protein throughout the virus life cycle (31); primarily, it complexes with the genomic RNA to form a ribonucleocapsid structure (17) and associates with the M protein (19, 39) to form the viral core (48). While N protein is required in trans to rescue the full-length clone of IBV (8) and a porcine coronavirus transmissible gastroenteritis virus clone (82), it is not required for others (1,71,72). Certainly, in the case of the rescue of the full-length clone of severe acute respiratory syndrome coronavirus, the presence of N protein increases viral titers compared to rescue performed in the absence of N transcript (83), suggesting that N protein may be involved in the efficiency of replication but that it is not essential.Based on amino acid sequence comparisons, three domains have been identified in the murine coronavirus, mouse hepatitis virus (MHV) N protein (46), of which the central domain (domain II) was identified as a potential RNA binding site (35, 40) capable of binding both coronavirus-and non-coronavirusderived RNA sequences (35,68). However, whether this binding occurs with equal or different affinity is uncertain (14,35,49). N protein has been shown to associate with several motifs on viral RNA, including the leader RNA sequence, with particular affinity for the core sequence of the TAS (2, 41), sequences at the 3Ј end of the genomic RNA (84), and the packaging signal (37). How these sequences promote N binding is unknown.Several coronavirus N proteins have been shown to be phosphorylated, including IBV, MHV, and transmissible gastroenteritis virus N proteins, although the precise sites were not identified (31). The role of phosphorylation in the virus life cycle is unknown, although the phosphorylation state of N protein has been predicted to pl...