Trafficking of Coronavirus Spike Protein may Play an Important Role in Cell-to-Cell Fusion

Sadasivan , Jibin (2016) Trafficking of Coronavirus Spike Protein may Play an Important Role in Cell-to-Cell Fusion. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Jibin S (11MS019)) MS_thesis_Jibin_S_11MS019_.pdf Restricted to Repository staff only Download (3MB)

Abstract

Cell-to-cell fusion is vital for intracellular viral spread. Neurotropic strain of Mouse hepatitis virus, MHV-A59 or its isogenic strain RSA59 is known to spread from grey matter to white matter via axonal transport and can infect oligodendrocytes and microglia by cell-to-cell fusion. By this intra-axonal transport, they can evade the immune response and cause white matter demyelinating disease by destructing oligodendrocytes. However a closely related strain RSMHV2 (isogenic strain of RSA59 except for spike gene), differ in the ability to spread from white matter to grey matter and successive cell-to-cell fusion. Spike gene plays a major role in intra-axonal transport and cell-to-cell fusion. A stretch of 16-26 high hydrophobic amino acids containing two consecutive central proline in RSA59 spike protein is predicted to play a major role in cell-to-cell fusion whereas RSMHV2 fusion peptide contains only one proline. Deletion of one proline in the fusion domain of RSA59 and addition of one proline in the fusion domain of RSMHV2 revealed that cis confirmation of two proline may be responsible for cell-to-cell fusion. Deletion of one proline from the fusion domain delays syncytia formation and reduced the viral titer. In vivo studies showed that the newly generated proline deleted recombinant strain is less neuropathogenic and slower in replication. Inorder to study whether two consecutive proline in the fusion peptide of RSA59 spike protein can exist in cis confirmation I expressed the peptide in bacterial system and purified by GST affinity chromatography. Although the proline deletion affects the fusogenecity and infectivity, it did not abolish the fusion completely. Therefore it is important to study the minimum essential motif of spike protein required for cell to cell fusion. Towards this goal we studied the fusion property of C terminal truncated spike proteins. Full length spike protein, when expressed exogenously is found to promote cell-to-cell fusion. Intracellular trafficking and localization studies of spike protein from MHV, SARS and OC43 showed that MHV spike protein is localized in the surface whereas SARS spike protein is localized in ER or ERGIC compartment and OC43 spike protein is predominantly localized in lysosome. Differential localization can be explained by signal sequence. The sequence alignment using clustal W shows that the signal sequence present at the cytoplasmic tail is playing important role in spike protein localization. We identified a unique GYQEL motif at the cytoplasmic terminal of OC43 spike protein which helps in localization in lysosome and a novel KLHYT motif in the cytoplasmic tail of SARS spike protein which helps in ER or ERGIC localization. The absence of signal sequence in the cytoplasmic tail of MHV-A59 spike protein is responsible for its surface localization and subsequent cell-to-cell fusion. My studies shed some light in understanding the role of spike protein in coronavirus pathogenesis. In long term this studies will help to develop novel mimetic peptide that can block the fusion and infection.

Actions (login required)

View Item