The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

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Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NP core ) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NP core exclude the N-and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NP core are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCEHantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. Hantavirus causes severe infectious diseases in human and animals (1, 2). In addition to 24 representative species, increasing numbers of antigenically and genetically distinct hantaviruses have recently been registered as new members of the genus Hantavirus (3, 4). The reservoir hosts of hantaviruses in nature are primarily rodents, shrews, moles, and bats, and the transmission of hantaviruses via aerosols of rodent excreta to humans causes two human diseases: (i) hemorrhagic fever with renal syndrome (HFRS), which is primarily caused by Hantaan virus (HTNV) in Asia, by Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe, and by Seoul virus (SEOV) worldwide, and ...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Guo

Wang

Sun

et al. 2016

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“…Using an ambisense coding strategy, the S segment encodes the nucleoprotein and a nonstructural protein (NSs). The nucleoprotein is a multifunctional protein that protects the viral genome, is involved in the replication/ transcription process, and has a role in virion assembly (3,4). The NSs protein of SFTSV has been shown to inhibit the type I interferon (IFN) response through sequestration of STAT proteins and other factors in viral replication complexes (5)(6)(7).…”

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confidence: 99%

Modeling Severe Fever with Thrombocytopenia Syndrome Virus Infection in Golden Syrian Hamsters: Importance of STAT2 in Preventing Disease and Effective Treatment with Favipiravir

Gowen

Westover

Miao

et al. 2017

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease endemic in parts of Asia. The etiologic agent, SFTS virus (SFTSV; family Bunyaviridae, genus Phlebovirus) has caused significant morbidity and mortality in China, South Korea, and Japan, with key features of disease being intense fever, thrombocytopenia, and leukopenia. Case fatality rates are estimated to be in the 30% range, and no antivirals or vaccines are approved for use for treatment and prevention of SFTS. There is evidence that in human cells, SFTSV sequesters STAT proteins in replication complexes, thereby inhibiting type I interferon signaling. Here, we demonstrate that hamsters devoid of functional STAT2 are highly susceptible to as few as 10 PFU of SFTSV, with animals generally succumbing within 5 to 6 days after subcutaneous challenge. The disease included marked thrombocytopenia and inflammatory disease characteristic of the condition in humans. Infectious virus titers were present in the blood and most tissues 3 days after virus challenge, and severe inflammatory lesions were found in the spleen and liver samples of SFTSV-infected hamsters. We also show that SFTSV infection in STAT2 knockout (KO) hamsters is responsive to favipiravir treatment, which protected all animals from lethal disease and reduced serum and tissue viral loads by 3 to 6 orders of magnitude. Taken together, our results provide additional insights into the pathogenesis of SFTSV infection and support the use of the newly described STAT2 KO hamster model for evaluation of promising antiviral therapies.IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease for which there are currently no therapeutic options or available vaccines. The causative agent, SFTS virus (SFTSV), is present in China, South Korea, and Japan, and infections requiring medical attention result in death in as many as 30% of the cases. Here, we describe a novel model of SFTS in hamsters genetically engineered to be deficient in a protein that helps protect humans and animals against viral infections. These hamsters were found to be susceptible to SFTSV and share disease features associated with the disease in humans. Importantly, we also show that SFTSV infection in hamsters can be effectively treated with a broad-spectrum antiviral drug approved for use in Japan. Our findings suggest that the new SFTS model will be an excellent resource to better understand SFTSV infection and disease as well as a valuable tool for evaluating promising antiviral drugs.

“…After years of NP structural investigation, several NSV nucleoprotein structures have been reported, such as Lassa fever virus (LAFV) (10,11), Crimean-Congo hemorrhagic fever virus (CCHFV) (12,13), influenza A virus (14), hantavirus (15), and SFTS virus (16) among segmented NSVs (sNSVs) and Borna disease virus (BDV) (17), vesicular stomatitis virus (VSV) (18,19), respiratory syncytial virus (RSV) (20), parainfluenza virus 5 (PIV-5) (21), and Nipah virus (NiV) (22) among nonsegmented NSVs. Despite the structural differences among these nucleoproteins, most of the structures share a typical bilobe fold consisting of N-and C-terminal domains, with a positive-charged RNA binding groove between the two lobes.…”

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confidence: 99%

Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 Peptide in Marburg Virus

Liu

Dong

et al. 2017

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Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NP core , both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NP core C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NP core remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus.IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously with self-assembly oligomerization. Here we report the structures of MARV NP core in two different forms. In the MARV NP apo form, we identify an interesting hexamer formed by hydrophobic interaction within a long helix, which is highly conserved and flexible among filoviruses and may indicate its critical function during the virus RNP formation. Moreover, the structural comparison with the NP-VP35 peptide complex reveals a structural transition chaperoned by VP35, in which the RNA binding groove undergoes a transition from closed state to open state. Finally, we discussed the high conservation and critical role of the VP35 binding pocket and its potential use for therapeutic development.KEYWORDS Marburg virus, nucleoprotein, crystal structure, filovirus, assembly mechanism