Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli.

Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM.

cis-Acting signals in encapsidation of Hantaan virus S-segment viral genomic RNA by its N protein.

The nucleocapsid (N) protein encapsidates both viral genomic RNA (vRNA) and the antigenomic RNA (cRNA), but not viral mRNA. Previous work has shown that the N protein has preference for vRNA, and this suggested the possibility of a cis-acting signal that could be used to initiate encapsidation for the S segment. To map the cis-acting determinants, several deletion RNA derivatives and synthetic oligoribonucleotides were constructed from the S segment of the Hantaan virus (HTNV) vRNA. N protein-RNA interactions were examined by UV cross-linking studies, filter-binding assays, and gel electrophoresis mobility shift assays to define the ability of each to bind HTNV N protein. The 5' end of the S-segment vRNA was observed to be necessary and sufficient for the binding reaction. Modeling of the 5' end of the vRNA revealed a possible stem-loop structure (SL) with a large single-stranded loop. We suggest that a specific interaction occurs between the N protein and sequences within this region to initiate encapsidation of the vRNAs.

Characterization of the Hantaan nucleocapsid protein-ribonucleic acid interaction.

The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K(d), for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg(2+) in these binding reactions had little impact on the K(d). However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg(2+). In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg(2+) or an increase in the ionic strength. The K(d) values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA.

Lymphocyte p56L32 is a RNA/DNA-binding protein which interacts with conserved elements of the murine L32 ribosomal protein mRNA.

In previous studies of the ribosomal protein L32 mRNA, we demonstrated that a conserved polypyrimidine tract found in the 5'-untranslated region (5'-UTR) was required for translational regulation in vivo and that a 56-kDa protein (p56L32) from T-lymphocytes specifically interacts with this sequence [Kaspar, R. L., Kakegawa, T., Cranston, H., Morris, D. R. & White, M. W. (1992) J. Biol. Chem. 267, 508-514]. Here we show that p56L32 binding to the L32 5'-UTR is complex and requires other 5'-UTR RNA sequences in conjunction with the polypyrimidine tract. Deletion and site-directed mutagenesis studies revealed that binding of p56L32 to the L32 5'-UTR requires a second RNA element, GGUGGCUGCC, 15 nucleotides downstream from the polypyrimidine tract. In contrast, L32 RNA transcripts altered in this downstream element were good substrates for binding of the polypyrimidine binding proteins from HeLa nuclear extracts, indicating that these proteins have RNA-binding specificities distinct from p56L32. Competition analysis demonstrated that p56L32 will bind to DNA as well as RNA with identical sequence specificity and similar affinity. Single or double-stranded DNAs composed of the L32 5'-UTR sequences were found to specifically compete with L32 RNA transcripts for p56L32 binding. The L32 5'-UTR downstream element, GGUGGCUGCC, which is required for p56L32 binding, has previously been implicated as a transcriptional element of the L32 gene. The ability of p56L32 to bind this sequence as DNA or RNA suggests p56L32 may have a dual role in the regulation of ribosomal protein mRNA accumulation and translation.