The Crimean-Congo Hemorrhagic Fever Virus NSm Protein is Dispensable for Growth In Vitro and Disease in Ifnar-/- Mice.
ABSTRACT: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tri-segmented, tick-borne nairovirus that causes disease of ranging severity in humans. The CCHFV M segment encodes a complex glycoprotein precursor (GPC) that undergoes extensive endoproteolytic cleavage, giving rise to two structural proteins (Gn and Gc) required for virus attachment and entry, and to multiple non-structural proteins (NSm, GP160, GP85, and GP38). The functions of these non-structural proteins remain largely unclear. Here, we investigate the role of NSm during infection by generating a recombinant CCHFV lacking the complete NSm domain (10200?NSm) and observing CCHFV ?NSm replication in cell lines and pathogenicity in Ifnar-/- mice. Our data demonstrate that the NSm domain is dispensable for viral replication in vitro, and, despite the delayed onset of clinical signs, CCHFV lacking this domain caused severe or lethal disease in infected mice.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.
Project description:Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5?Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a ?-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.
Project description:Nairoviruses are primarily tick-borne bunyaviruses, some of which are known to cause mild-to-severe febrile illness in humans or livestock. We describe the genome sequences of 11 poorly characterized nairoviruses that have ecological associations with either birds (Farallon, Punta Salinas, Sapphire II, Zirqa, Avalon, Clo Mor, Taggert, and Abu Hammad viruses), rodents (Qalyub and Bandia viruses), or camels (Dera Ghazi Khan virus). Global phylogenetic analyses of proteins encoded in the L, M, and S RNA segments of these and 20 other available nairovirus genomes identified nine well-supported genogroups (Nairobi sheep disease, Thiafora, Sakhalin, Keterah, Qalyub, Kasokero, Dera Ghazi Khan, Hughes, and Tamdy). Genogroup-specific structural variations were evident, particularly in the M segment encoding a polyprotein from which virion envelope glycoproteins (Gn and Gc) are generated by proteolytic processing. Structural variations include the extension, abbreviation, or absence sequences encoding an O-glycosylated mucin-like protein in the N-terminal domain, distinctive patterns of conserved cysteine residues in the GP38-like domain, insertion of sequences encoding a double-membrane-spanning protein (NSm) between the Gn and Gc domains, and the presence of an alternative long open reading frame encoding a viroporin-like transmembrane protein (Gx). We also observed strong genogroup-specific associations with categories of hosts and tick vectors.
Project description:Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.
Project description:Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.
Project description:The genus Nairovirus of arthropod-borne bunyaviruses includes the important emerging human pathogen, Crimean-Congo hemorrhagic fever virus (CCHFV), as well as Nairobi sheep disease virus and many other poorly described viruses isolated from mammals, birds, and ticks. Here, we report genome sequence analysis of six nairoviruses: Thiafora virus (TFAV) that was isolated from a shrew in Senegal; Yogue (YOGV), Kasokero (KKOV), and Gossas (GOSV) viruses isolated from bats in Senegal and Uganda; Issyk-Kul virus (IKV) isolated from bats in Kyrgyzstan; and Keterah virus (KTRV) isolated from ticks infesting a bat in Malaysia. The S, M, and L genome segments of each virus were found to encode proteins corresponding to the nucleoprotein, polyglycoprotein, and polymerase protein of CCHFV. However, as observed in Leopards Hill virus (LPHV) and Erve virus (ERVV), polyglycoproteins encoded in the M segment lack sequences encoding the double-membrane-spanning CCHFV NSm protein. Amino acid sequence identities, complement-fixation tests, and phylogenetic analysis indicated that these viruses cluster into three groups comprising KKOV, YOGV, and LPHV from bats of the suborder Yingochiroptera; KTRV, IKV, and GOSV from bats of the suborder Yangochiroptera; and TFAV and ERVV from shrews (Soricomorpha: Soricidae). This reflects clade-specific host and vector associations that extend across the genus.
Project description:UNLABELLED:The nairoviruses include assorted tick-borne bunyaviruses that are emerging as causative agents of infectious diseases among humans and animals. As negative-sense single-stranded RNA (-ssRNA) viruses, nairoviruses encode nucleoprotein (NP) that encapsidates the genomic RNA and further forms ribonucleoprotein (RNP) complex with viral RNA-dependent RNA polymerase (RdRp). We previously revealed that the monomeric NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV) presents a racket-shaped structure and shows unusual DNA-specific endonuclease activity. To examine the structural and biological variation of nairovirus-encoded NPs, here, we systematically solved the crystal structures of NPs encoded by various nairoviruses, including Hazara virus (HAZV), Kupe virus (KUPV), and Erve virus (ERVEV). Combined with biochemical analysis, our results generate a clearer picture to aid in the understanding of the functional diversity of nairovirus-encoded NPs and the formation of nairovirus RNPs. IMPORTANCE:Nairoviruses comprise several tick-borne bunyaviruses that are emerging as causative agents of infectious diseases among humans and animals; however, little is known of the nairovirus genome assembly and transcription mechanisms. Based on the previous study of CCHFV NP reported by different research groups, we systematically investigate here the structural and functional diversity among three different nairoviruses. This work provides important information on nairovirus nucleoprotein function and the formation of RNPs.
Project description:Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm', was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm' was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts.