RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment

Abstract

The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negativesense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5 terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5-terminal 32 and 3-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 g of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5 terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.Bunyamwera virus (BUN) is the prototype of the genus Bunyavirus and the family Bunyaviridae and possesses a singlestranded negative-sense tripartite RNA genome. The three RNA segments, termed L (large), M (medium), and S (small), encode six proteins. The L segment codes for the L protein, the viral RNA-dependent RNA polymerase, which is responsible for both transcribing and replicating the genome RNAs. The M segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, as a polyprotein which is probably cotranslationally cleaved by host proteases. The S segment encodes the nucleocapsid (N) protein and, in an overlapping reading frame, a second nonstructural protein called NSs (reviewed in reference 8).In common with other negative-sense RNA viruses, the bunyavirus genome RNA segments are replicated via full-length cRNAs termed antigenomes. Both the negative-sense genome and positive-sense antigenome RNAs are encapsidated by the N protein and are associated with the viral polymerase in ribonucleoprotein complexes called nucleocapsids. It is only within the nucleocapsid that the RNA is transcriptionally active. Bunyavirus genome and antigenome RNAs contain highly conserved, complementary terminal sequences that may form panhandle structures in vivo and are probably responsible for the circular appearance of isolated nucleocapsids (19,20,22,26,28).Full-length genome and antigenome segments are usually the only RNAs that are encapsidated in the infected cell. Viral mRNAs, which are not encapsidated, are truncated at the 3Ј end and contain a nontemplated capped primer on the 5Ј terminus (2, 4, 9, 14, 21). It is therefore likely that the terminal sequences of the genome and antigenome RNAs are involved in the encapsidation process. This theory is supported by the observation that an antisense chloramphenicol acetyltransferas...

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

“…Previous studies based on extraction and sequencing of bacterial RNA bound by LEAV N's homolog, BUNV N, during expression in bacterial cells seem to rule out sequence specific binding of RNA by BUNV N (23,24). Perhaps the specificity could be limited to weak or tight binding, giving rise to regions of RNA that could be preferentially or easily uncapsidated for initiation of events such as replication or transcription (35)(36)(37). Viral RNA is encapsidated by N proteins resulting in the formation of RNP complexes.…”

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein

“…For LACV, since the RNA binding to the core does not involve significant conformational changes, we imagine that polymerase-induced changes in arm conformation, which as we have shown is coupled to RNA conformation, are most likely used to temporarily disrupt RNA binding. Indeed it is possible that the same kind of interaction of the NP arms with L protein, perhaps also involving specific RNA sequence recognition (36), enables recruitment of auto-inhibited NP into a conformation able to lock onto nascent genome replicates during de novo RNP assembly.…”

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

“…Bunyavirus N binds single-stranded RNA (ssRNA) nonspecifically (6, 7), although some N may have a preference for specific viral RNA sequences (8)(9)(10). Studies on some animal viruses within the Bunyaviridae family found that encapsidated RNA is resistant to disruption by high salt and Ribonuclease treatment (8,10,11).…”

“…Studies on some animal viruses within the Bunyaviridae family found that encapsidated RNA is resistant to disruption by high salt and Ribonuclease treatment (8,10,11). Despite the common property of tight, nonspecific binding to single-stranded RNA, homology of N within the Bunyaviridae family is not apparent from sequence data, because N from different genera appear unrelated.…”