Overriding the complement barrier: The ups and downs in the Rhabdovirus's battle for survival

Abstract

Background: Rhabdoviruses are non-segmented, negative sense RNA viruses and are potent pathogens causing infection in humans and animals, e.g., rabies virus, Chandipura virus. Vesicular stomatitis virus (VSV) is a prototypic rhabdovirus. The complement system is a potent barrier against an array of pathogens including VSV. Complement activation by VSV involves the classical pathway resulting in virus neutralization. However, VSV signature responsible for complement activation is unknown. Being the only surface glycoprotein, we investigated the role of G protein in complement activation. We also investigated the mechanism of modulation of human complement by VSV which included the recruitment of the host membrane associated regulators of complement activation (RCA). Methods and materials: Cell surface activation of complement by VSV was carried out by infecting Vero cells with wt-VSV or transfecting with VSV-G plasmid (pCAGGS-G) and treating with normal human serum followed by immunofluorescence microscopy and flow cytometry. G protein from the virion and infected cells was purified by affinity-chromatography and validated using silver staining. Complement binding and activation by the purified protein was tested using multiple biochemical approaches. The specificity of RCA modulation by VSV was assessed by performing time course experiments in VSV infected HeLa cells followed by western blotting and RT-PCR. RCA incorporation and their relative function in virion budding out at specific time points were validated by cofactor and decay accelerating activity. Results: Immunofluorescence and flow cytometric analysis of VSV infected and G transfected cells showed significant deposition of active complement proteins C3b and C4b suggesting a role for the G protein. Interaction of VSV-G with C3/C4 components of complement was further confirmed by ELISA. Fluid phase activation assays with purified G confirmed the role of G in binding to and activating complement proteins, further supported by SPR studies. Western blotting, FACS experiments with infected HeLa cells showed preference of CD55 over CD46 in complement modulation by VSV. The inactivation of C3b and decay acceleration of C3 convertase by the virion associated CD46 and CD55 conferred VSV with resistance against complement. Conclusion: The VSV-G protein plays a significant role in complement activation pathways. Acknowledgement: Effective modulation of complement neutralization is supported by the RCA CD55.