Mohammad A. Mir

Hantavirus nucleocapsid protein has distinct m7G cap and RNA binding sites

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.

Signatures of Host mRNA 5′ Terminus for Efficient Hantavirus Cap Snatching

ABSTRACT Hantaviruses, similarly to other negative-strand segmented RNA viruses, initiate the synthesis of translation-competent capped mRNAs by a unique cap-snatching mechanism. Hantavirus nucleocapsid protein (N) binds to host mRNA caps and requires four nucleotides adjacent to the 5′ cap for high-affinity binding. N protects the 5′ caps of cellular transcripts from degradation by the cellular decapping machinery. The rescued 5′ capped mRNA fragments are stored in cellular P bodies by N, which are later efficiently used as primers by the hantaviral RNA-dependent RNA polymerase (RdRp) for transcription initiation. We showed that N also protects the host mRNA caps in P-body-deficient cells. However, the rescued caps were not effectively used by the hantavirus RdRp during transcription initiation, suggesting that caps stored in cellular P bodies by N are preferred for cap snatching. We examined the characteristics of the 5′ terminus of a capped test mRNA to delineate the minimum requirements for a capped transcript to serve as an efficient cap donor during hantavirus cap snatching. We showed that hantavirus RdRp preferentially snatches caps from the nonsense mRNAs compared to mRNAs engaged in translation. Hantavirus RdRp preferentially cleaves the cap donor mRNA at a G residue located 14 nucleotides downstream of the 5′ cap. The sequence complementarity between the 3′ terminus of viral genomic RNA and the nucleotides located in the vicinity of the cleavage site of the cap donor mRNA favors cap snatching. Our results show that hantavirus RdRp snatches caps from viral mRNAs. However, the negligible cap-donating efficiency of wild-type mRNAs in comparison to nonsense mRNAs suggests that viral mRNAs will not be efficiently used for cap snatching during viral infection due to their continuous engagement in protein synthesis. Our results suggest that efficiency of an mRNA to donate caps for viral mRNA synthesis is primarily regulated at the translational level.

Characterization of the interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19)

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.

Interaction of Hantavirus Nucleocapsid Protein with Ribosomal Protein S19

ABSTRACT Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that initiate the translation of their capped mRNAs by a novel mechanism mediated by viral nucleocapsid protein (N). N specifically binds to the mRNA 5′ m7G cap and 40S ribosomal subunit, a complex of 18S rRNA and multiple ribosomal proteins. Here, we show that N specifically interacts with the ribosomal protein S19 (RPS19), located at the head region of the 40S subunit. We suggest that this N-RPS19 interaction facilitates ribosome loading on capped mRNAs during N-mediated translation initiation.

The Triplet Repeats of the Sin Nombre Hantavirus 5′ Untranslated Region Are Sufficient in cis for Nucleocapsid-Mediated Translation Initiation

ABSTRACT Hantavirus nucleocapsid protein (N) can replace the cellular cap-binding complex, eukaryotic initiation factor 4F (eIF4F), to mediate translation initiation. Although N can augment translation initiation of nonviral mRNA, initiation of viral mRNA by N is superior. All members of the Bunyaviridae family, including the species of the hantavirus genus, express either three or four primary mRNAs from their tripartite negative-sense genomes. The 5′ ends of the mRNAs contain nonviral heterologous oligonucleotides that originate from endonucleolytic cleavage of cellular mRNA during the process of cap snatching. In the hantaviruses these caps terminate with a 3′ G residue followed by nucleotides arising from the viral template. Further, the 5′ untranslated region (UTR) of viral mRNA uniformly contains, near the 5′ end, either two or three copies of the triplet repeat sequence, UAGUAG or UAGUAGUAG. Through analysis of a panel of mutants with mutations in the viral UTR, we found that the sequence GUAGUAG is sufficient for preferential N-mediated translation initiation and for high-affinity binding of N to the UTR. This heptanucleotide sequence is present in viral mRNA containing either two or three copies of the triplet repeat.

Bunyavirus N: eIF4F surrogate and cap-guardian

Hantaviruses comprise a genus of the bunyavirus family of viruses. Viruses of this family, along with the arenaviruses, and the orthomyxoviruses, including influenza, contain a negative sense, segmented RNA genome. Viral nucleocapsid proteins play a well-established role in the formation of intracellular and virion-associated nucleocapsids that harbor and shield viral genomic RNA. However, recent observations indicate that hantavirus nucleocapsid protein (N) has additional unexpected biological activities that interface with both the cellular mRNA translation and mRNA degradation apparatus. N has an activity that mimics or circumvents the cellular cap-binding complex, eIF4F, in the initial stages of translation initiation. As a consequence of its translation initiation activity, N can augment translational expression. In addition to its ability to enhance translation initiation, N co-localizes with the cellular peptides that mediate mRNA decay. mRNA decay often takes place in cytoplasmic processing bodies (P-bodies), and N is abundant in P bodies. The association of N with P bodies enables cap-snatching for viral transcription initiation. It is likely that these two surprising new activities of N function in concert during bunyavirus gene expression. All the activities of N revolve around the ability of N to recognize RNA in a correct, context-dependent manner.

Recent Advances in Hantavirus Molecular Biology and Disease

Abstract Hantaviruses are emerging zoonotic pathogens that belong to the Bunyaviridae family. They have been classified as category A pathogens by CDC (centers for disease control and prevention). Hantaviruses pose a serious threat to human health because their infection causes two highly fatal diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These pathogens are transmitted to humans through aerosolized excreta of their infected rodent hosts. Hantaviruses have a tripartite-segmented negative-sense RNA genome. The three genomic RNA segments, S, M, and L, encode a nucleocapsid protein (N), a precursor glycoprotein that is processed into two envelope glycoproteins (Gn and Gc) and the viral RNA-dependent RNA polymerase (RdRp), respectively. N protein is the major structural component of the virus, its main function is to protect and encapsidate the three genomic RNAs forming three viral ribonucleocapsids. Recent studies have proposed that N in conjunction with RdRp plays important roles in the transcription and replication of viral genome. In addition, N preferentially facilitates the translation of viral mRNA in cells. Glycoproteins, Gn and Gc, play major roles in viral attachment and entry to the host cells, virulence, and assembly and packaging of new virions in infected cells. RdRp functions as RNA replicase and transcriptase to replicate and transcribe the viral RNA and is also thought to have endonuclease activity. Currently, no antiviral therapy or vaccine is available for the treatment of hantavirus-associated diseases. Understanding the molecular details of hantavirus life cycle will help in the identification of targets for antiviral therapeutics and in the design of potential antiviral drug for the treatment of HFRS and HCPS. Due to the alarming fatality of hantavirus diseases, development of an effective vaccine against hantaviruses is a necessity.

Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis

ABSTRACT Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.

Targeting a Novel RNA-Protein Interaction for Therapeutic Intervention of Hantavirus Disease

An evolutionarily conserved sequence at the 5′ terminus of hantaviral genomic RNA plays an important role in viral transcription initiation and packaging of the viral genome into viral nucleocapsids. Interaction of viral nucleocapsid protein (N) with this conserved sequence facilitates mRNA translation by a unique N-mediated translation strategy. Whereas this evolutionarily conserved sequence facilitates virus replication with the assistance of N in eukaryotic hosts having multifaceted antiviral defense, we demonstrate its interaction with N presents a novel target for therapeutic intervention of hantavirus disease. Using a high throughput screening approach, we identified three lead inhibitors that bind and induce structural perturbations in N. The inhibitors interrupt N-RNA interaction and abrogate both viral genomic RNA synthesis and N-mediated translation strategy without affecting the canonical translation machinery of the host cell. The inhibitors are well tolerated by cells and inhibit hantavirus replication with the same potency as ribavarin, a commercially available antiviral. We report the identification of a unique chemical scaffold that disrupts a critical RNA-protein interaction in hantaviruses and holds promise for the development of the first anti-hantaviral therapeutic with broad spectrum antiviral activity.

Andes virus nucleocapsid protein interrupts protein kinase R dimerization to counteract host interference in viral protein synthesis.

ABSTRACT Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. IMPORTANCE Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.