doi: 10.1074/jbc.M110.102459. 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 2,3-DCPE hydrochloride cellular chaperones of the HSP70 family CDC18L was confirmed during live CCHFV contamination. Using infectious HAZV, we showed for the first time that this nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is usually 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 usually 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 usually 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 this 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 1,000-fold, suggesting that this interaction is usually 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. INTRODUCTION The family of trisegmented negative-sense RNA viruses comprises five genera, namely, (1). The genus contains several serogroups, one of which is the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup, with single members CCHFV and the genetically distinct Hazara virus (HAZV) (2) that are formally grouped under the same species name of CCHFV. CCHFV is usually a risk group 4 human pathogen, responsible for a devastating disease for which preventative or therapeutic measures do not 2,3-DCPE hydrochloride exist (3). Transmission of CCHFV to humans 2,3-DCPE hydrochloride often occurs by the bite of infected ixodid ticks of the genus (4), and the human case-fatality rate can exceed 60% (5). In recent years, the occurrence of CCHFV-mediated disease has been newly reported in many Mediterranean countries (3), likely as a consequence of the increasingly broad habitat and population size of its tick vector, with the increases possibly occurring in response to climate change (6). CCHFV is now recognized as a potential threat to human health in the densely populated regions of Northern Europe (7). In contrast, HAZV has not been associated with serious human disease and is classified as a risk group 2 pathogen. HAZV contamination of type 1 interferon receptor-deficient mice shares clinical features of CCHFV-mediated disease in humans and represents an accessible CCHFV contamination model (8). Taken together, these findings suggest that HAZV is usually 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. Such nairoviruses include Erve virus, which causes thunderclap headaches in humans (9), and Nairobi 2,3-DCPE hydrochloride sheep disease virus, which is responsible for hemorrhagic gastroenteritis in livestock such as sheep and goats (10). The nairovirus genome comprises three strands of negative-sense RNA that are named small (S), medium (M), and large (L), reflecting their relative sizes. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the envelope glycoproteins Gn and Gc, and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). As with all bunyaviruses, 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 usually 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 formation of the nairovirus RNP is also dependent on the ability of the N protein to interact with itself to form.