Fibrinogen Gamma Chain Promotes Aggregation of Vesicular Stomatitis Virus in Saliva

Abstract

The spread of viruses among cells and hosts often involves multi-virion structures. For instance, virions can form aggregates that allow for the co-delivery of multiple genome copies to the same cell from a single infectious unit. Previously, we showed that vesicular stomatitis virus (VSV), an enveloped, negative-strand RNA virus, undergoes strong aggregation in the presence of saliva from certain individuals. However, the molecular components responsible for such aggregation remain unknown. Here we show that saliva-driven aggregation is protein dependent, and we use comparative proteomics to analyze the protein content of strongly versus poorly aggregating saliva. Quantitative analysis of over 300 proteins led to the identification of 18 upregulated proteins in strongly aggregating saliva. One of these proteins, the fibrinogen gamma chain, was verified experimentally as a factor promoting VSV aggregation in a dose-dependent manner. This study hence identifies a protein responsible for saliva-driven VSV aggregation. Yet, the possible involvement of additional proteins or factors cannot be discarded.

Keywords: collective infectious units; fibrinogen; label-free proteomics; vesicular stomatitis virus; viral transmission; virion aggregation.

Figure 1

Effect of saliva on vesicular stomatitis virus (VSV) aggregation as determined by foci analysis. (A) Fluorescence microscopy images of BHK-21 cells inoculated with VSV-mCherry and VSV-eGFP. A 1:1 mix of these two viruses was incubated for 1h at 37 °C with PBS or human saliva from donor P6, used for inoculating cells, and images were taken at 13 hpi. Bars indicate the percentage of foci positive for eGFP only (green), mCherry only (red), and doubly fluorescent foci (purple). Error bars represent the SEM from three technical replicates. (B) Effect of trypsin on saliva-driven VSV aggregation. Prior to inoculation, the 1:1 mix of VSV-mCherry and VSV-eGFP was incubated for 1 h at 37 °C in PBS, in saliva from donor P8, in P8 saliva previously mixed with FBS (Saliva_F), or in P8 saliva treated with trypsin (2 h at 37 °C) followed by trypsin inactivation with FBS (Saliva_T_F). Error bars represent the SEM from three technical replicates.

Figure 2

Volcano plots showing differential proteins expression. (A) Group 1 versus Group 2. Red dots: proteins upregulated in Group 2. Blue dots: proteins downregulated in Group 2. Selection criteria were (i) that abundance should differ by at least twofold in Groups 2 and 1 and that (ii) differences in abundance should be significant at the 0.05 level in a t-test adjusted for a 5% FDR after multiple testing (q-value < 0.05). (B) Group 3 versus Group 2. Red dots: proteins showing statistically indistinguishable or greater expression level in Group 3 compared to Group 2. Only proteins that were measurable in all 6 samples were used for this comparison. A List of all proteins is available from Table S2.

Figure 3

Fibrinogen gamma-chain (FGG) effect on vesicular stomatitis virus (VSV) aggregation assayed by flow cytometry. Top: Scatter plots (>100,000 events) showing non-infected (blue), mCherry-positive (red), eGFP-positive (green), and doubly fluorescent (purple) cells at 6 hpi in cultures inoculated with a 1:1 mix VSV-mCherry VSV-eGFP (0.1 PFU/cell). Prior to inoculation, the mix was either incubated (1 h, 37 °C) in saliva from donor P7 or in P7 saliva supplemented with recombinant FGG (125 ng/mL). Scatter plot data show one out of the three technical replicates conducted. Bottom: histograms showing the mean percentage of foci positive for eGFP only (green), mCherry only (red), and doubly fluorescent foci (purple) from three technical replicates (error bars: SEM).

Figure 4

Relationship between viral aggregation and fibrinogen levels. (A) Green and purple bars represent ELISA-determined fibrinogen levels in saliva from Group 1 and Group 2 donors (error bar: SEM). (B) Correlation between fibrinogen concentration and aggregation, as determined by foci co-fluorescence analysis. The line shows the least-squares regression fit.