Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

Abstract

Measles virus (MV) is a negative-strand RNA virus within the Morbillivirus genus of the Paramyxoviridae family. The MV transcriptional complex consists of the virus-encoded RNAdependent RNA polymerase (L), the polymerase cofactor (P), and a ribonucleoprotein template consisting of the singlestranded RNA genome and the nucleocapsid protein (N protein). N protein monomers assemble on the RNA genome during replication to form a single-start left-handed helix, protecting the genome from nuclease degradation. Encapsidation is initiated on specific sequences found within the leader RNA, drawing upon pools of soluble N-P heterodimers, whereas elongation of encapsidation occurs in an RNA sequence-independent manner (7, 24). Since genomic replication presumably is dependent upon concurrent encapsidation, functional motifs in the N protein required for genomic replication must include an RNA binding domain for the initiation of encapsidation, a binding site for P to form an N-P encapsidation complex, and N-N interaction sites required to drive nucleocapsid elongation and maintain nucleocapsid structural integrity. Additionally, N protein must contain a P binding site that is exposed on the nucleocapsid, thereby permitting the viral polymerase complex to interact with formed ribonucleoprotein templates during both transcription and genomic replication.The expression of MV N protein deletion mutants demonstrates that only the amino-terminal three-fourths of the molecule (i.e., amino acids 1 to 398) is required for the formation of organized nucleocapsid-like particles, localizing the N-N interaction domain to this highly conserved portion of the protein (1, 16). This conclusion is supported by the observation that selective proteolysis of the MV nucleocapsid can remove the C-terminal 15 kDa of the N protein while leaving nucleocapsid structural integrity and the amino-terminal 45-kDa N protein fragment intact (12). Also identified within the latter region are two sites necessary for the formation of soluble N-P complexes, localized to amino acids 4 to 188 and 304 to 373 (1). Enhanced sensitivity of the C terminus of the N protein to proteolysis is consistent with its exposure on the surface of the formed nucleocapsid. Within this exposed hypervariable domain is a third binding site for P, tentatively localized between amino acids 457 and 525 (1). For Sendai virus, the C-terminal domain of the N protein is required for template function in RNA replication assays (5) and contains a P binding site (4). Given the essential role of P in supporting viral polymerase function, a model for paramyxoviruses emerges in which P provides the link between L and N protein domains that are exposed on the nucleocapsid and that are necessary for both transcription and replication.The putative template function of the MV N protein C

Identification of potential HLA class I and class II epitope precursors associated with heat shock protein 70 (HSPA)

“…In our report, the HSPA-associated peptides revealed a hydrophobic arrangement, interspersed with hydrophilic residues, including acidic ones, and we did not find preferences for aromatic residues (Table 1). Therefore, our data, with reference to the HSPA binding motif, are in agreement with the in vivo study of Grossmann et al (2004), but are in contrast to HSPA binding motifs which have been proposed in the in vitro studies (Fourie et al 1994;Maeda et al 2007;Takenaka et al 1995;Zhang et al 2002). The binding of the peptides co-purified with HSPA was tested with recombinant HSPA1A and HSPA8 on a peptide array.…”

“…Therefore, in order to gain further insight into the profile of the HSPA-associated peptides, we studied the preferential binding motif within the peptides. It has been reported that HSPA preferentially binds peptides with a hydrophobic core (Fourie et al 1994;Grossmann et al 2004;Maeda et al 2007;Takenaka et al 1995), with preference for basic (Fourie et al 1994;Grossmann et al 2004;Maeda et al 2007;Takenaka et al 1995) and large aromatic residues (Fourie et al 1994), and that acidic amino acids are rarely present (Fourie et al 1994;Maeda et al 2007;Zhang et al 2002). In our report, the HSPA-associated peptides revealed a hydrophobic arrangement, interspersed with hydrophilic residues, including acidic ones, and we did not find preferences for aromatic residues (Table 1).…”

The C-terminal Domain of the Measles Virus Nucleoprotein Is Intrinsically Disordered and Folds upon Binding to the C-terminal Moiety of the Phosphoprotein

“…Purification of PNT and of N were carried out as described in (25,48). The 43-kDa N-terminal fragment of N, N CORE , was obtained by limited trypsin digestion of N as described in Ref.…”

“…The 43-kDa N-terminal fragment of N, N CORE , was obtained by limited trypsin digestion of N as described in Ref. 25, followed by gel filtration onto a Superdex 200 HR 10/30 column (Amersham Biosciences). N CORE was eluted with 50 mM Tris/HCl, pH 7.5, 1 mM PMSF and stored at Ϫ20°C in the presence of 20% glycerol.…”

“…In Morbilliviruses and Respiroviruses, the region responsible for binding of nucleocapsids to P is located within N TAIL (13,18,20,24). Consequently, N TAIL is required for N⅐RNA to act as a template for viral RNA synthe-sis (13,16,25). N TAIL has also been shown to be required for stable binding of N°to P (16,26).…”