GP38 is a structural component of Crimean-Congo hemorrhagic fever virus particles

Highlights

• •LC-MS/MS analysis indicated comparable levels of GP38 and viral structural proteins (NP, Gn, Gc) in purified CCHFV particles.
• •Western blot and IEM results confirm the presence of GP38 in CCHFV particles.
• •CCHFV particles were imaged and viral morphology was characterized the first time.

Dear Editor,

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe viral hemorrhagic fever epidemics with a case fatality rate of up to 40%, and is a World Health Organization (WHO) priority pathogen. CCHFV, belonging to the family Nairoviridae in the class Bunyaviricetes, has a segmented negative-single-strand RNA genome, which is named as large (L), medium (M) and small (S) segment according to their size. The M segment encodes the glycoprotein precursor (GPC), and the GPC is further cleaved into five proteins, including N-terminal mucin like domain (MLD), glycoprotein 38 (GP38), glycoprotein N (Gn), nonstructural protein m (NSm), and glycoprotein C (Gc) (Hawman and Feldmann, 2023). Gn and Gc anchor in the virion membrane via their C-terminal transmembrane regions. As a class II membrane fusion protein (Li et al., 2022; Mishra et al., 2022), Gc mediates the membrane fusion between viral membrane and host cell membrane, while Gn may act as a chaperone protein to cover the fusion loop of Gc and control where and when the fusion occurs (Guardado-Calvo and Rey, 2021). GP38 was detected as a secreted glycoprotein of 38 kDa in the supernatant of the infected cells, and was not considered as a structural protein (Sanchez et al., 2006). Studies on the human immune response to natural CCHFV infection have demonstrated that the main response antibodies target Gc and GP38, with most neutralizing antibodies against Gc (Fels et al., 2021). Some neutralizing antibodies against Gc demonstrated protection efficacy against CCHFV infection in mouse models (Mishra et al., 2022; Li et al., 2024). Interestingly, although antibodies against GP38 cannot neutralize CCHFV in vitro, a few provided protection against lethal challenge with CCHFV in a mouse model (Golden et al., 2019). Recently, secreted GP38 was found to cause vascular leak and viral spread, which partially explained the protective function of the non-neutralizing anti-GP38 antibody (Pahmeier et al., 2025). Additionally, Golden et al. showed that the protection of the non-neutralizing anti-GP38 antibody requires complement C3, and postulated that GP38 may be associated with CCHFV particles or infected cells to mediate the complement function (Golden et al., 2019). Their data showed that GP38 was associated with CCHFV viral like particles (VLPs) and cells transfected with plasmid containing M segment (Golden et al., 2019). There is a hint that GP38 may be a structural component of CCHFV particles from recent publications. For example, Guardado-Calvo and Rey pointed out that from a structural point of view, the heterodimer formed by Gn and GP38 of CCHFV shared evolutionary commonality with chaperone proteins of other class II membrane fusion proteins (Guardado-Calvo and Rey, 2021). The high-resolution cryo-EM structure of engineered GP38-Gn-Gc protein provided a potential molecular basis for GP38's association with the virion (McFadden et al., 2024). However, so far there is lack of direct evidence that GP38 is indeed associated with CCHFV particles. Here, we aimed to obtain direct evidence of the presence of GP38 on the virions by analyzing the purified CCHFV particles through a series of techniques.

We first purified CCHFV particles from infected BSR-T7/5 cells (please see Supplementary Materials for detailed methods). Briefly, the supernatant of the infected cells was fixed with 4% paraformaldehyde for inactivation, and the virions were purified with sucrose dense-gradient ultracentrifugation. Purified CCHFV particles were observed by transmission electron microscopy (TEM). Negative staining EM revealed near-spherical CCHFV particles, and the surface of the virions was covered with a dense layer of envelope protein (Fig. 1A). To better visualize the intact native CCHFV particles, cryo-electron microscopy (cryo-EM) was used. The morphology of the virus particles observed by cryo-EM was mostly spherical or ovoid (Fig. 1B). The diameter of spherical particles ranged from 80 to 140 nm (Fig. 1C). The short axis of ovoid particles was mainly distributed around 90 nm, and the long axis ranged from 105 to 165 nm (Fig. 1D).

Fig. 1.

Multiple analyses confirmed that GP38 located in CCHFV particles collected from infected BSR-T7/5 cells. A Purified CCHFV particles were negative stained and examined by electron microscopy. Scale bar: 100 nm. B A micrograph of frozen-hydrated CCHFV particles. Scale bar: 100 nm. C Histogram of diameters for spherical CCHFV particles. The diameters of spherical viral particles (n = 46) were measured from cryo-EM images. D Histogram of long and short axes for ovoid CCHFV particles (n = 23) measured from cryo-EM images. E Mass spectrometry analysis results of virion proteins. Purified CCHFV particles were analyzed by LC-MS/MS as described in Methods. iBAQ: intensity based absolute quantification. F Western blot analysis of viral proteins on virions. Purified CCHFV particles were analyzed by 12% SDS–polyacrylamide gel electrophoresis (PAGE), and mouse monoclonal antibodies against CCHFV NP, Gn, Gc and GP38 were used as indicated. G Silver staining of 10% SDS-PAGE gel revealed specific bands corresponding to Gc, NP, Gn, and GP38. H Immuno-electron microscope analysis of CCHFV. Purified CCHFV particles were incubated with the indicated antibodies and stained with 12 nm gold particle conjugated secondary antibodies. Samples were then negative stained and examined by electron microscopy. mAb-Gc, mAb-Gn, and mAb-GP38 were monoclonal antibodies against CCHFV Gc, Gn, and GP38, respectively. mAb-SFTSV-Gn: irrelative monoclonal antibody targeting SFTSV Gn as negative control. Scale bar: 100 nm.

We then investigated whether GP38 is associated with CCHFV particles with a series of techniques. First, purified CCHFV particles were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). GP38 was identified as a major virion component. Its intensity-Based Absolute Quantification (iBAQ) value, which correlates with molar protein quantity, was comparable to those of Gn, Gc and NP (all ≥ 2.93 × 10⁹) (Fig. 1E and Table 1). In contrast, the iBAQ values of other proteins were significantly lower (RdRp: 2.56 × 10⁷; NSm: 1.56 × 10⁸). The high abundance of GP38 detected in the CCHFV particles indicated that the presence of GP38 was not due to non-specific adsorption but was most likely as a structural component of the virions.

Table 1.

Mass spectrometry analysis of virion proteins.

Protein ID	Length	Mw (kDa)	Virions purified from BSR-T7/5 cells				Virions purified from Huh-7 cells
			Unique peptides	Unique peptide coverage [%]	Intensity	iBAQa	Unique peptides	Unique peptide coverage [%]	Intensity	iBAQa
NP	482	53.89	53	91.9	2.71 × 1011	9.69 × 109	40	79	2.21 × 1010	7.90 × 108
Gn	288	32.25	19	63.5	4.69 × 1010	2.93 × 109	14	43.8	1.97 × 109	1.31 × 108
Gc	693	78.06	41	62.5	1.01 × 1011	2.98 × 109	24	44.3	1.05 × 1010	2.84 × 108
GP38	272	30.74	23	69.5	5.92 × 1010	4.55 × 109	16	47.8	3.03 × 109	2.33 × 108
RdRP	3945	447.94	105	38.1	5.40 × 109	2.56 × 107	55	18.2	5.39 × 108	2.55 × 106
NSm	234	25.95	1	12.4	1.56 × 109	1.56 × 108	1	12.4	4.08 × 106	4.08 × 105

^aiBAQ: intensity-based absolute quantification.

Second, purified CCHFV particles were detected by Western blotting with monoclonal antibodies against NP, Gn, Gc, and GP38 respectively (Fig. 1F and Supplementary Table S1). The reported main viral proteins could be detected as specific protein bands with their expected sizes (NP: ∼55 kDa, Gn: ∼40 kDa, Gc: ∼78 kDa). As expected, antibody against GP38 showed strong reactivity to the 38-kDa protein. In addition to GP38, weaker signals were also detected between 70-kDa and 100-kDa protein marker bands, which may correspond to uncleaved GP38-Gn or GP85. During GPC processing, GP85 was generated in the ER, which was further cleaved by furin to generate GP38 and MLD at Golgi (Sanchez et al., 2006). Due to the incomplete cleavage by furin, GP85 was found secreted in the supernatant (Sanchez et al., 2006). However, further experiments are needed to characterize these bands. Silver staining of the purified virion sample on an SDS-PAGE gel also revealed specific bands corresponding to Gc, NP, Gn, and GP38 (Fig. 1G).

Third, CCHFV particles were analyzed by immune-electron microscopy (IEM). To this end, CCHFV particles were incubated with monoclonal antibody (mAb) against Gc, Gn, or GP38. An irrelevant monoclonal antibody targeting severe fever with thrombocytopenia syndrome virus (SFTSV) Gn protein was used as negative control. Purified CCHFV particles were then stained with 12 nm gold particles conjugated secondary antibodies and were negatively stained for TEM examination. When mAb-Gn, mAb-Gc, and mAb-GP38 were used as primary antibodies, gold particles were specifically detected around the virus particles. When an irrelative primary antibody was used in control experiments, gold particles were found in the background but were almost absent on virions (Fig. 1H, full size images in Supplementary Fig. S1). Therefore, like Gn and Gc, GP38 is also specifically detected in the CCHFV particles.

The above data were generated from CCHFV particles purified from infected BSR-T7/5 cells. Given that the liver is the main target of CCHFV in vivo, we purified CCHFV particles from infected Huh-7 (Human hepatocellular carcinoma cell line) cells (Fig. 2A), and repeated the above detections. Our results showed that similar to that of BSR-T7/5 cells, GP38 was detected on the virion particles by LC-MS/MS, Western blotting, and IEM (Fig. 2B–D).

Fig. 2.

Multiple analyses confirmed that GP38 located in CCHFV particles collected from infected Huh-7 cells. A Purified CCHFV particles were negatively stained and examined by electron microscopy. Scale bar: 100 nm. B Mass spectrometry analysis results of virion proteins. Purified CCHFV particles were analyzed by LC-MS/MS as described in Methods. iBAQ: intensity based absolute quantification. C Western blot analysis of viral proteins on virions. Purified CCHFV particles were analyzed by 12% SDS-polyacrylamide gel electrophoresis, and mouse monoclonal antibodies against CCHFV NP, Gn, Gc and GP38 were used as indicated. D Immuno-electron microscope analysis of CCHFV. Purified CCHFV particles were incubated with the indicated antibodies and stained with 12 nm gold particle conjugated secondary antibodies. Samples were then negative stained and examined by electron microscopy. mAb-Gc, mAb-Gn, and mAb-GP38 were monoclonal antibodies against CCHFV Gc, Gn, and GP38, respectively. mAb-SFTSV-Gn: irrelative monoclonal antibody targeting SFTSV Gn as negative control. Scale bar: 100 nm.

In conclusion, we demonstrated the existence of GP38 on the CCHFV particles by LC-MS/MS, Western blotting, and IEM. Our finding is important for CCHFV studies in several respects. First, it provides direct evidence to support the current structure model that GP38-Gn-Gc on the surface of CCHFV (McFadden et al., 2024). Second, it opens a few critical questions regarding the role of GP38 in CCHFV infection. For example, what is the function of GP38 on the virions? As suggested before, the presence of GP38 on the surface of virus particles may stabilize the pre-fusion conformation of Gc, so that no membrane fusion occurs during the release of viral particles from infected cells (McFadden et al., 2024). Whether the presence of GP38 on the virion could trigger antibody mediated complement activation, and thus contribute to the protection of non-neutralizing anti-GP38 antibodies as previously suggested (Golden et al., 2019)? It has been found that the secreted GP38 of CCHFV impairs endothelial barrier function and causes vascular leak, which leads to increased viral dissemination (Pahmeier et al., 2025). How does the virus regulate the distribution of secreted GP38 and structural-related GP38? And finally, our study also sheds new light on the design of prevention strategy against CCHFV.

Footnotes

We thank Mrs. Qinghong Xiao, Prof. Bing Yan from Wuhan Institute of Virology for their work on preparation of monoclonal antibodies against Gn, GP38, and Gc; Prof. Fei Deng form Wuhan Institute of Virology for providing monoclonal antibodies against CCHFV-NP; Prof. Leike Zhang from Wuhan Institute of Virology for providing BSR-T7/5 cells; Mr. Jia Wu, Mr. Hao Tang, Mr. Jun Liu, and Mr. Tao Du from BSL-3 Laboratory; Mr. Ding Gao, Mrs. Pei Zhang, Mrs. Bichao Xu and Mrs. Mingyu Wei from the Core Facility and Technical Support of Wuhan Institute of Virology, CAS for their critical support. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0490000), the National Natural Science Foundation of China (U22A20336), and the National Key R&D Program of China (2022YFC2303300; 2023YFC2305900). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Prof. Manli Wang is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Figure S1.

The original full-size images of the immunogold staining and electron microscopy of CCHFV. The dotted boxes in A indicate the position of the particles shown in Fig. 1H, and boxes in B indicate the area shown in Fig. 2D. mAb-Gc, mAb-Gn, and mAb-GP38 were monoclonal antibodies against CCHFV Gc, Gn, and GP38, respectively. mAb-SFTSV-Gn: irrelative monoclonal antibody targeting SFTSV Gn as negative control. Scale bar: 100 nm.