Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

Abstract

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di-and trimeric forms. The mono-and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono-and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3 and 5 ends of the genomic RNA. In contrast, the mono-and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

Bunyavirus N: eIF4F surrogate and cap-guardian

“…Separate or overlapping domains of N are likely to be involved in cap-recognition and panhandle recognition, required for cap-snatching/translation and genome encapsidation, respectively. The panhandle is recognized with specificity and high affinity by N. 69 As discussed in the text, N also binds to 5' caps of cellular mRNAs through its eIF4E-like activity. Cleavage of the bound cellular mRNA, presumably by the polymerase, generates a capped oligonucleotide.…”

Characterization of the Interaction between Hantavirus Nucleocapsid Protein (N) and Ribosomal Protein S19 (RPS19)

“…First, a T7 terminator was excised from pSNV N plasmid using NotI and SphI restriction sites and cloned between the same sites in a PUC19 vector to generate the pUCT7ter plasmid. Second, the gene encoding the viral S segment mRNA with appropriate 5Ј and 3Ј UTR sequences was PCR-amplified from pADS vector (27) using the forward primer (5Ј-ATTATATCCCGGGTAATACGACTCACTATA-GGAGTGAGTGTAGTAGTAGACTCCTTGAGAAGCTAC-TACGACTAAAG) and the reverse primer (5Ј-ATTATTAGC-GGCCGCCGTCTCATTATATACCCTGTAATATAATTG-GCCCTG). The forward primer with flanking T7 promoter contained the XmaI restriction site, and the reverse primer contained the NotI and BsmBI restrictions sites.…”

“…Multiple in vitro studies have shown that N preferentially binds viral RNA compared with complementary RNA or nonviral RNA (14,(21)(22)(23)(24)(25)(26). It has been proposed that the specific binding of N with either the panhandle or the sequence at the 5Ј terminus alone selectively facilitates the encapsidation of viral RNA to generate three nucleocapsids that are packaged into infectious virions (26,27). The RNA binding domain of Hantaan virus N has been mapped to the central conserved region corresponding to amino acid residues 175-217(25).…”

Hantavirus Nucleocapsid Protein Has Distinct m7G Cap- and RNA-binding Sites

“…RNA Synthesis by T7 Transcription Reaction-DNA encoding the full-length S segment RNA molecule with a proximally located T7 promoter was generated by PCR using an upstream primer containing a T7 promoter sequence, a downstream primer that overlaps the terminal sequence of the S segment transcription unit, and a pAD-S plasmid as template, as described previously (26). The resulting DNA containing the entire S segment transcription unit was gel-purified and used directly in transcription reactions with T7 RNA polymerase (MBI Fermentase).…”

“…Multiple in vitro studies have shown that N preferentially binds vRNA compared with complementary RNA (cRNA) or nonviral RNA (13, 20 -25). It has been proposed that the specific binding of N with either the panhandle or the sequence at the 5Ј terminus alone selectively facilitates the encapsidation of vRNA to generate three nucleocapsids that are packaged into infectious virions (25,26). The RNA-binding domain of Hantaan virus N protein has been mapped to the central conserved region corresponding to amino acids from 175 to 217 (24).…”