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

Abstract

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...

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

“…Although cap-binding activity has not been directly demonstrated (8,20), it is tempting to speculate that N may replace eIF4E in recruiting eIF4G and eIG4A to viral mRNAs. Indeed, the hantavirus cap-binding nucleocapsid protein has been shown to interact directly with the small ribosomal protein RPS19 to bypass the eIF4F complex entirely (10,19).…”

Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

“…Recently, peroxiredoxin 1 was identified as a binding partner of the nucleocapsid protein of measles virus and was found to be required for its replication and transcription (63). Hantavirus nucleocapsid protein binds to a ribosomal protein, RPS19, and mediates preferential translation of viral mRNA (14). Thus, several viral replication proteins appear to interact with cellular proteins to modulate a variety of functions required for successful replication of the virus in the host.…”

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

“…One interesting example of a viral protein interacting with host ribosomal proteins is the case of the hantavirus nucleocapsid (N) protein, which interacts with the RPS19 protein (47)(48)(49). It has been shown that RPS19 interaction with N protein facilitates ribosome loading onto the viral RNA transcripts, leading to preferential translation of viral transcripts.…”