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ABSTRACT: The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.
Proceedings of the National Academy of Sciences 01/2009; 105(49):19294-9. · 9.68 Impact Factor
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ABSTRACT: A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.
Journal of Virology 12/2006; 80(22):11283-92. · 5.40 Impact Factor
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ABSTRACT: Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein, encoded by the smallest of the three genome segments (S), has nonspecific RNA chaperone activity. This activity results in transient dissociation of misfolded RNA structures, may be required for facilitating correct higher-order RNA structure, and may function in viral genome replication. We carried out a series of experiments to further characterize the ability of N to dissociate RNA duplexes. As might be expected, N dissociated RNA duplexes but not DNA duplexes or RNA-DNA heteroduplexes. The RNA-destabilizing activity of N is ATP independent, has a pH optimum of 7.5, and has an Mg(2+) concentration optimum of 1 to 2 mM. N protein is unable to unwind the RNA duplexes that are completely double stranded. However, in the presence of an adjoining single-stranded region, helix unwinding takes place in the 3'-to-5' direction through an unknown mechanism. The N protein trimer specifically recognizes and unwinds the terminal panhandle structure in the viral RNA and remains associated with unwound 5' terminus. We suggest that hantaviral nucleocapsid protein has an active role in hantaviral replication by working cooperatively with viral RNA polymerase. After specific recognition of the panhandle structure by N protein, the unwound 5' terminus likely remains transiently bound to N protein, creating an opportunity for the viral polymerase to initiate transcription at the accessible 3' terminus.
Journal of Virology 08/2006; 80(13):6276-85. · 5.40 Impact Factor
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ABSTRACT: Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.
Journal of Virology 03/2005; 79(3):1824-35. · 5.40 Impact Factor
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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.
Journal of Virology 09/2004; 78(15):8281-8. · 5.40 Impact Factor
