Detection of receptor-induced glycoprotein conformational changes on enveloped virions by using confocal micro-Raman spectroscopy

Abstract

Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.

Fig 1

Raman spectroscopy signals of NiV VLPs. (A) Electron micrograph of NiV VLPs obtained from 293T cells expressing NiV-M, -F, and -G glycoproteins. The NiV-F and -G spikes are marked by black arrows. (B) Western blot analysis of total proteins in NiV VLPs. Briefly, VLPs generated by 293T cells were denatured and detected with a rabbit anti-AU1 monoclonal antibody against AU1-tagged NiV-F (top), a mouse anti-flag monoclonal antibody against flag-tagged NiV-M (middle), and a mouse anti-HA monoclonal antibody against HA-tagged NiV-G (bottom). One of three representative experiments is shown. (C) Raman spectral features of NiV VLPs. The spectrum in red is PCDNA3 (negative control), and spectra in black, green, and blue are three independently produced NiV VLP samples generated in 293T cells. (D to F) Principal component analysis (PCA) validates the significant variations of Raman spectra of different types of NiV VLPs (i.e., MFG, MF, MG, and M) produced in various cell lines: 293T cells (D), Vero cells (E), and CHO cells (F). The relative locations of the signals on the plot are a result of the relative concentrations of the sources of each signal (e.g., F, G, and M, in each total sample) and do not correlate with the identity of the signal. Therefore, the locations in the PCA plot will vary for the specific protein signals among viral preparations (for example, from distinct cell types). The parameter that measures the distinctiveness between signals is the Mahalanobis distance between given signal groups (explained in Materials and Methods). Data points are circled to simplify their identification. Red, MPCDNA3; blue, MF; black, MG; green, MFG.

Fig 2

Raman spectral features of M, F, and G proteins on NiV VLPs cultivated in 3 different cell lines. (A to C) The Raman spectral signals for M, F, and G glycoproteins were obtained from mathematical subtraction of the respective Raman signals. For example, the F signals were obtained from subtracting the MG signals from the MFG signals and by subtracting the M signals from the MF signals. Similarly, the G signals were obtained by subtracting the MF signals from the MFG signals and by subtracting the M signals from the MG signals. Comparison of the two types of subtractions was performed and yielded nearly identical patterns (D_y1y2 value of less than 300, indicating high spectral reproducibility). NiV VLPs were produced from 293T cells (A), Vero cells (B), and CHO cells (C). (D) Average of virus-like particle signals produced from three distinct cell cultures. Calculation of spectral reproducibility (D_y1y2 value of <650) showed no significantly different signals derived from the 3 cell lines. (E) NiV-G and NiV-F signals were obtained similarly as described above for panels A to C but by subtracting NiV-G/VSV from VSV bald-particle Raman signals or NiV-F/VSV from VSV bald-particle Raman signals. At least 3 independent experiments were performed to gather each Raman spectrum.

Fig 3

Second-derivative transformation analyses of Raman spectral features reveal clear specific M, F, and G spectral peaks. (A) Second-derivative transformation of Raman spectral features (1,500 to 1,200 cm⁻¹) of M, F, and G proteins on NiV VLPs (from the Vero cell line). (B) Second-derivative transformation of Raman spectral features (1,200 to 900 cm⁻¹) of M, F, and G proteins on NiV VLPs (from the Vero cell line). (C) A representative PCA classification model was established and cross-validated to differentiate NiV proteins (n = 4). Data points are circled to simplify their identification. Blue, F protein; black, G protein; red, M protein.

Fig 4

Energy input results in the detection of G and F glycoprotein conformational variations in NiV/VSV pseudovirions. (A) F glycoprotein conformational variations were monitored by using the featured Raman bands at wave numbers of 1,409 cm⁻¹ and 1,302 cm⁻¹ at different time intervals at room temperature (25°C). Different colors denote selective time intervals. (B) G glycoprotein conformational variations were monitored by using the featured Raman bands at wave numbers of 1,337 cm⁻¹ and 1,321 cm⁻¹ at different time intervals at room temperature (25°C). Different colors denote selective time intervals. (C and D) Raman spectroscopy-based 2D correlation synchronous plot as a function of spectral wave number for F glycoprotein (C) and G glycoprotein (D). Gray shading indicates negative cross-peaks, and no shading indicates positive cross-peaks (n = 5).

Fig 5

Raman spectroscopy detects a receptor-induced conformational change in NiV-F in pseudotyped virions. (A) Conformational variation of the NiV-G glycoprotein in pseudovirions prebound to ephrinB2 receptor, prior to or after a 3-min incubation at 25°C. The NiV-G spectroscopic signal changes correlate with the temperature-induced conformational changes observed for NiV-G in Fig. 4B. (B) Conformational variation and stability of the NiV-F glycoprotein in viral particles prebound to ephrinB2 receptor, prior to or after a 3-min incubation at 25°C. The Raman signals on the 1,409-cm⁻¹ peak of NiV pseudovirions switched to the opposite orientation from those induced by temperature for the same 1,409-cm⁻¹ peak in NiV-F in Fig. 4A, indicating that Raman spectroscopy can detect a receptor-induced conformational change in NiV-F. (C) Similar experiment as in panel A, except that it was performed with viral particles containing the G fusion mutant 4-5 (13). This mutant is capable of receptor binding but incapable of triggering F to induce membrane fusion. (D) Similar experiment as in panel B, except that it was performed with viral particles containing the G fusion mutant 4-5 (13) (n = 5).

Fig 6

A receptor-induced conformational change in NiV-F can occur at 25°C and depends on the presence of a target membrane. (A) F-triggering assay. Briefly, CHO cells transfected with NiV-F, NiV-G, and GFP were mixed with receptor-containing PK13B2 cells at 4°C for 90 min. Subsequently, cell mixtures were incubated at 37°C, 25°C, or 4°C for 60 min in the presence or absence of biotinylated HR2 peptide, and the amount of HR2 binding was calculated at each temperature. In the experiment labeled 37°C, sol B2, soluble ephrinB2-Fc replaced PK13B2 cells. The averages of data from 3 experiments ± standard deviations are shown. (B) Conformational variation of NiV-G upon binding to soluble ephrinB2. Shown is an experiment similar to that in Fig. 5A, except that it was performed in the presence of soluble ephrinB2-Fc instead of ephrinB2/VSV reversed pseudotyped virions. (C) Conformational variation of NiV-F upon NiV/VSV pseudovirion binding to soluble ephrinB2. Shown is an experiment similar to that in Fig. 5B, except that it was performed in the presence of soluble ephrinB2-Fc instead of ephrinB2/VSV reversed pseudotyped virions (n = 3).