Identification and characterization of inhibitors for hantavirus replication

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DESCRIPTION (provided by applicant): Hantaviruses, members of the Bunyaviridae family are negative stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodents. The hantaviral genome is composed of three negative sense genomic RNA segments: S, M and L that encode nucleocapsid protein (N), glycoprotein precursor (GPC) and viral RNA dependent RNA polymerase (RdRp), respectively. The GPC is cleaved in the middle, generating an N-terminal fragment (Gn) and a C-terminal fragment (Gc). The glycoprotein Gn harbors a cytoplasmic tail domain of 142 amino acids at the C-terminus. Hantaviruses have evolved a novel translation initiation mechanism, operated by N, which preferentially favors the translation of viral mRNAs in the host cels. N binds to the ribosomal protein S19 (RPS19), a structural component of 40S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and 5' UTR favors ribosome loading on viral transcripts during translation initiation. There is growing evidence that other negative stranded RNA viruses such as influenza use similar mechanisms for the translation of their mRNAs. We have developed a tractable assay to study the interaction of N with mRNA cap and viral mRNA 5' UTR. We would like to develop this assay for screening chemical libraries in high throughput mode for the identification of molecules that inhibit N-cap and N-UTR interaction. N protein also plays a key role in encapsidation and packaging of viral genome. All minus stranded, segmented RNA viruses have genome segments that are found in "panhandle" conformation via the interaction of the genome termini. We have found that panhandle structure is the primary high affinity binding substrate for hantavirus N. We have strong preliminary data showing that N-panhandle complex specifically interacts with the cytoplasmic tail domain of glycoprotein Gn. Our results demonstrate that binding of N to the viral RNA (vRNA) panhandle generates a novel nucleoprotein complex that selectively targets vRNA for encapsidation. The specific interaction between N-panhandle complex and Gn cytoplasmic tail domain selectively transports the nucleocapsids to specific destinations on Golgi membranes that are studded with the glycoproteins. Thus, the specific interaction between Gn cytoplasmic tail domain and N-panhandle complex likely mediates the selective incorporation of vRNA derived nucleocapsids into virions. We have developed a fluorescence based assay to study the interaction between N-panhandle complex and Gn cytoplasmic tail domain. We will upgrade this assay for the identification of molecules that interfere in the interaction between N-panhandle complex and Gn tail domain. In future, these assays will be used for screen larger chemical libraries for the identification of molecules that inhibit the replication of a broad spectrum of negative stranded RNA viruses, including medically important viruses such as hantaviruses, influenza virus etc. ! PUBLIC HEALTH RELEVANCE: Hantavirus cardio pulmonary syndrome (HPS) cased by rodent born category A Sin Nombre hantavirus has a mortality of fifty percent. There is no treatment for hantavirus associated disease at present. Development of high throughput screening assays for the identification of molecules that inhibit transcription, translation and packaging of hantaviral genome will reveal inhibitors that will be used to prevent humans from hantavirus infection.