Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

Severson, William; Xu, Xiaolin; Kühn, Michaela; Senutovitch, Nina; Thokala, Mercy; Ferrón, François; Longhi, Sonia; Canard, Bruno; Jonsson, Colleen B.

doi:10.1128/jvi.79.15.10032-10039.2005

Cited by 42 publications

(38 citation statements)

References 39 publications

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“…The K app (apparent equilibrium constant ϭ 1/K d ) was calculated at four different temperatures (8,15,25, and 30°C) by fluorometric titration of 1,5-IAEDANSlabeled RPS19 with N. ⌬H and ⌬S were calculated from the slope and intercept of a plot of ln K app against 1/T (Equation 4). ⌬G was calculated from Equation 5 after incorporation of the values for ⌬H and ⌬S obtained from Equation 4.…”

Section: Methodsmentioning

confidence: 99%

“…During encapsidation, N specifically recognizes the three viral RNA genome segments inside the host cell and targets them for packaging. 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).…”

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confidence: 99%

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

Cheng

Haque

Rimmer

et al. 2011

Journal of Biological Chemistry

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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 rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5 cap and a highly conserved triplet repeat sequence of the viral mRNA 5 UTR. The simultaneous binding of N at both the terminal cap and the 5 UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.Hantaviruses, members of the Bunyaviridae family, are category A pathogens and causative agents of two emerging diseases: hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome with mortalities of 15 and 50%, respectively (1, 2). Hantaviruses are transmitted to humans through aerosolized excreta of infected rodent hosts. The spherical hantavirus particles harbor three negative sense genomic RNA segments (S, L, and M) within a lipid bilayer (3). The mRNAs derived from S, L, and M segments encode viral nucleocapsid protein (N), 2 viral RNA-dependent RNA polymerase (RdRp), and glycoprotein precursor, respectively. The glycoprotein precursor is cleaved at a conserved WAASA site, and two glycoproteins, Gn and Gc, are generated (4). The characteristic feature of the hantaviral genome is the partially complementary sequence at the 5Ј and 3Ј termini of each of the three genome segments that undergo base pairing and form panhandle structures (5-7). N is a multifunctional protein playing vital roles in multiple processes of the virus replication cycle and enters the host cell along with viral capsid during infection. N has been found to undergo trimerization both in vivo and in vitro (8 -20). During encapsidation, N specifically recogniz...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Cheng

Haque

Rimmer

et al. 2011

Journal of Biological Chemistry

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“…This observation verifies the presence of different binding sites on N for m 7 G capped decamer and viral RNA. Hantaviruses have a highly conserved RNA binding domain located at 175-217 amino acids (24). Deletion of this RNAbinding domain abrogates the ability of N to interact with viral S segment RNA (24,54).…”

Section: Purification Of Hantavirus Nucleocapsid Protein-e Colimentioning

confidence: 99%

“…Hantaviruses have a highly conserved RNA binding domain located at 175-217 amino acids (24). Deletion of this RNAbinding domain abrogates the ability of N to interact with viral S segment RNA (24,54). We generated a deletion mutant of N that lacks the RNA binding domain and was purified as described in "Experimental Procedures" (Fig.…”

Section: Purification Of Hantavirus Nucleocapsid Protein-e Colimentioning

confidence: 99%

“…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).…”

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

Mir

Sheema

Haseeb

et al. 2010

Journal of Biological Chemistry

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Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that carry three negative stranded RNA molecules as their genome. Hantavirus nucleocapsid protein (N) is encoded by the smallest S segment genomic RNA (viral RNA). N specifically binds mRNA caps and requires four nucleotides adjacent to the cap for high affinity binding. We show that the N peptide has distinct cap-and RNA-binding sites that independently interact with mRNA cap and viral genomic RNA, respectively. In addition, N can simultaneously bind with both mRNA cap and vRNA. N undergoes distinct conformational changes after binding with either mRNA cap or vRNA or both mRNA cap and vRNA simultaneously. Hantavirus RNA-dependent RNA polymerase (RdRp) uses a capped RNA primer for transcription initiation. The capped RNA primer is generated from host cell mRNA by the cap-snatching mechanism and is supposed to anneal with the 3 terminus of vRNA template during transcription initiation by single G-C base pairing. We show that the capped RNA primer binds at the cap-binding site and induces a conformational change in N. The conformationally altered N with a capped primer loaded at the cap-binding site specifically binds the conserved 3 nine nucleotides of vRNA and assists the bound primer to anneal at the 3 terminus. We suggest that the cap-binding site of N, in conjunction with RdRp, plays a key role during the transcription and replication initiation of vRNA genome.Hantaviruses cause two types of serious human illnesses when transmitted to humans from rodent hosts: hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome (1, 2). The spherical hantavirus particles harbor three negative sense genomic RNA segments (S, L, and M segments) within a lipid bilayer (3). The mRNAs derived from S, L, and M segments encode viral nucleocapsid protein (N), viral RNA-dependent RNA polymerase (RdRp), 2 and glycoproteins (G1 and G2), respectively. The characteristic feature of the hantaviral genome is the partially complementary sequence at the 5Ј and 3Ј termini of each of the three genome segments that undergo base pairing and form panhandle structures (4 -6). N is a multifunctional protein playing a vital role in multiple processes of virus replication cycle and has been found to undergo trimerization both in vivo and in vitro (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). During encapsidation, the three viral RNA (vRNA) molecules are specifically recognized by N inside the host cell and targeted for packaging. 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 f...

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Intrinsic Flexibility of Nucleic Acid Chaperone Proteins from Pathogenic RNA Viruses

Ivanyi‐Nagy

Makowska

Darlix

2012

Protein and Peptide Folding, Misfolding, and Non‐Folding

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Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

Cited by 42 publications

References 39 publications

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

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

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

Intrinsic Flexibility of Nucleic Acid Chaperone Proteins from Pathogenic RNA Viruses

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