Abstract
ABSTRACTThe Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease.IMPORTANCE Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance.
Highlights
Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease
To identify cellular proteins that interacted with the nairovirus N protein during the replication cycle, an IP-based strategy was used to isolate the Crimean-Congo hemorrhagic fever virus (CCHFV) N protein from cells in culture followed by mass spectrometry (MS) to identify coprecipitating cellular proteins
We utilized both quantitative proteomics and conventional IP protocols to elucidate the identity of interacting cellular proteins that coprecipitate with CCHFV and Hazara virus (HAZV) N proteins during both the intracellular and extracellular phases of their replication cycles
Summary
Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. HAZV infection of type 1 interferon receptor-deficient mice shares clinical features of CCHFV-mediated disease in humans and represents an accessible CCHFV infection model [8] Taken together, these findings suggest that HAZV is a useful surrogate that can be used to study the molecular, cellular, and disease biology of the highly pathogenic CCHFV, as well as of other nairoviruses responsible for serious human and animal diseases. The nairovirus genome and antigenome are encapsidated by multiple copies of the viral N protein to form a ribonucleoprotein (RNP) complex This N-RNA association is thought to be critical for the virus replication cycle, and only in the form of the RNP can the CCHFV genome be transcribed and replicated [11]. The functional consequences of these interactions within the virus replication cycle have not been fully elucidated, those studies showed that N was capable of interacting with cellular proteins
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