Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle

Abstract

The 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.

FIG 1

Expression and immunoprecipitation of EGFP–CCHFV-N in HEK293T cells. (A) HEK293T cells were transfected with a plasmid designed to express an EGFP–CCHFV-N fusion protein, which was detected by Western blot analysis of cell lysates using an anti-CCHFV N antibody. M, molecular mass marker lane (values at the left are in kilodaltons). (B) HEK293T cells were transfected with plasmids expressing EGFP or EGFP–CCHFV-N, and their subcellular localization was determined using confocal microscopy, with the nucleus stained blue using DAPI and with EGFP and EGFP–CCHFV-N detected as green. (C and D) Following GFP-trap-mediated IP from EGFP- and EGFP–CCHFV-N-expressing cells, proteins were resolved by SDS-PAGE and visualized by either Coomassie staining (C) or Western blotting (D) with an anti-GFP antibody. Unfilled arrowheads denote EGFP–CCHFV-N.

FIG 2

Validation of selected CCHFV N protein cellular interaction partners. HEK293T cells were transfected with EGFP–CCHFV-N and EGFP, and, 24 h later, cell lysate components were immunoprecipitated with GFP-trap, RFP-trap, or unconjugated agarose beads. The resulting IPs were separated by PAGE and then subjected to Western blotting to probe for the presence of the selected cellular proteins listed using specific antibodies. The ratios of the abundances of these proteins determined by the two SILAC LC-MS/MS analyses are indicated, and a complete list of all interaction partners is provided as an annotated table in Data set S1 in the supplemental material.

FIG 3

Interaction between cellular HSP70 and native CCHFV N expressed transiently or during CCHFV infection. (A) Anti-CCHFV N antisera was used for IP of native CCHFV N expressed in HEK293T cells, and the isolated proteins were analyzed by Western blotting using the antibodies shown. (B) The subcellular localization of native CCHFV N in HUH-7 cells was investigated using indirect immunofluorescence microscopy using anti-CCHFV N antisera, shown as green. Nuclei are stained with DAPI, shown as blue. (C) Anti-CCHFV N antisera was used for IP of native CCHFV N from CCHFV-infected SW13 cells, and the isolated proteins were analyzed by Western blotting using the antibodies shown. (D) The subcellular localization of native CCHFV N in CCHFV-infected SW13 cells was investigated by indirect immune fluorescence microscopy using anti-CCHFV N antisera, shown as green. The nuclei are stained with DAPI, shown as blue. (E) Indirect confocal immunofluorescence analysis of SW13 cells transiently expressing native CCHFV-N protein expressed from a plasmid cDNA. Cells were costained using antibodies specific for CCHFV N (green) and anti-HSP70 (red), with donkey and chicken secondary antibodies, respectively, as well as DAPI (blue).

FIG 4

Analysis of the interaction between HAZV N and HSP70 in cells. (A) HAZV N was immunoprecipitated from HAZV-infected cells using anti-N antisera and then subjected to Western blotting using the antibodies shown. (B) Indirect confocal immunofluorescence analysis of SW13 cells infected with HAZV revealed HAZV N (green) localized in discrete cytoplasmic puncta, similarly to CCHFV N. HSP70 (red) was present in both the cytoplasm and nucleus and also colocalized with HAZV N in some cytoplasmic puncta. Nuclei are stained with DAPI, shown as blue. Bottom row shows mock-infected cells.

FIG 5

SDS-PAGE analysis of purified HAZV harvested from SW13 cell supernatant and Western blot confirmation of the presence of HSP70 and HSP90 family members. (A and B) Secreted HAZV particles harvested from infected SW13 cell supernatants were purified by PEG precipitation followed by centrifugation through an iodixanol gradient. HAZV particles were isolated (lane VF1), and the remaining 4 fractions (1 ml each) were collected from the top of the gradient (Fraction 2) to the bottom of the gradient (Fraction 5). Fractions were analyzed by SDS-PAGE and either Coomassie stained (A) or silver stained (B). (C) Fraction VF1 was subjected to Western blot analysis using both anti-HAZV N antisera and antibodies targeting the listed cellular proteins, including HSP70. (D) HAZV N was immunoprecipitated from purified infectious HAZV and then subjected to Western blot analysis using anti-HAZV N and anti-HSP70 antibodies.

FIG 6

The effect of HSP70/HSC70 inhibitors VER and PIF on HAZV replication. (A) Cytotoxicity of VER and PIF in SW13 cells was assessed by MTT assay after 24 h of incubation of SW13 cells with VER and PIF at several different concentrations in the absence of HAZV. Cytotoxicity levels are presented relative to 0.4% DMSO results. (B) The effect on HAZV replication of treating A549 cells with VER and PIF was determined by plaque assay, which revealed a reduction in HAZV titers at subcytotoxic PIF and VER concentrations. The statistical significance of decreased virus multiplication using PIF and VER in relation to DMSO-only controls was assessed using paired Student's t tests, and significance is shown with an asterisk (* [P < 0.05]). (C) Western blot analysis of HAZV N protein and GAPDH levels within infected cells following PIF and VER treatment at subcytotoxic concentrations. (D) HAZV N protein levels quantified from Western blot analyses performed in three experiments.