For positive-strand RNA viruses, genomic RNA (gRNA) functions both as a message for translation and as a template for the viral RNA-dependent RNA polymerase. As part of their infectious cycle, these viruses often extensively remodel host cellular membranous compartments to facilitate replication and transcription of gRNA. These expropriated membranes are thought to provide surfaces for the concentration of enzymatic and substrate components of RNA synthesis, to furnish general protection from cellular ribonucleases, and to allow evasion of host innate immunity through the sequestration of double-stranded viral RNA intermediates. Among all known RNA viruses, coronaviruses have the largest genomes, ranging from 27 to 32 kb, and the most complex machinery for RNA synthesis (21,35,74). The coronavirus replicase-transcriptase complex (here called the replicase) is assembled from 16 nonstructural proteins (nsp), which collectively total some 803 kDa for mouse hepatitis virus (MHV). The nsp are autoproteolytically processed from two overlapping polyprotein precursors, which are produced by translation of gRNA. Almost all of the replicase components are associated with or embedded in convoluted membranes and double-membrane vesicles that are generated during coronavirus infection (29).In addition to the replicase, the nucleocapsid protein (N) is known to play an important role in coronavirus RNA synthesis, although that role is, as yet, poorly defined (1,51,60,76). Multiple investigations have shown intracellular colocalization of N with replicase components at early stages of infection by MHV or by severe acute respiratory syndrome coronavirus (SARS-CoV) (14,55,58,61). The participation of N protein in an early event in RNA synthesis is also implied by the poor adherence of coronaviruses to the nearly universal rule that the gRNA of positive-strand RNA viruses is completely infectious when introduced into susceptible host cells. Some time ago, we noted that the infectivity of purified MHV gRNA was markedly enhanced by cotransfection of synthetic N mRNA (36); this phenomenon was rediscovered when full-length reversegenetic systems were devised for coronaviruses. For systems that are launched by transfection of gRNA, the provision of cotransfected N mRNA or N-encoding plasmid either is greatly stimulatory (10,(69)(70)(71) or is absolutely required (6) for the recovery of infectious virus.In the present study, we analyzed the consequences of replacing the MHV N open reading frame (ORF) with its counterpart from the closely related bovine coronavirus (BCoV). This substitution resulted in the discovery of a previously unsuspected genetic interaction between the serine-and argininerich (SR) region of the N protein and a region of the nsp3 subunit of the viral replicase. We confirmed this interaction through biochemical pulldown experiments. Moreover, we found that there is a correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of MHV gRNA. Our results offer an explanation for t...

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Human leukocyte antigen (HLA)-DQ2.5 (**) is a class-II major histocompatibility complex protein associated with both type 1 diabetes and celiac disease. One unusual feature of DQ2.5 is its high class-II-associated invariant chain peptide (CLIP) content. Moreover, HLA-DQ2.5 preferentially binds the non-canonical CLIP2 over the canonical CLIP1. To better understand the structural basis of HLA-DQ2.5's unusual CLIP association characteristics, better insight into the HLA-DQ2.5·CLIP complex structures is required. To this end, we determined the X-ray crystal structure of the HLA-DQ2.5· CLIP1 and HLA-DQ2.5·CLIP2 complexes at 2.73 and 2.20 Å, respectively. We found that HLA-DQ2.5 has an unusually large P4 pocket and a positively charged peptide-binding groove that together promote preferential binding of CLIP2 over CLIP1. An α9-α22-α24-α31-β86-β90 hydrogen bond network located at the bottom of the peptide-binding groove, spanning from the P1 to P4 pockets, renders the residues in this region relatively immobile. This hydrogen bond network, along with a deletion mutation at α53, may lead to HLA-DM insensitivity in HLA-DQ2.5. A molecular dynamics simulation experiment reported here and recent biochemical studies by others support this hypothesis. The diminished HLA-DM sensitivity is the likely reason for the CLIP-rich phenotype of HLA-DQ2.5.

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