A proline insertion-deletion in the core of fusion peptide of mouse hepatitis virus spike glycoprotein alters structural rigidity and modulates neuropathogenesis

Singh, Manmeet (2019) A proline insertion-deletion in the core of fusion peptide of mouse hepatitis virus spike glycoprotein alters structural rigidity and modulates neuropathogenesis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Manmeet Singh (13RS039)) 13RS039.pdf - Submitted Version Restricted to Repository staff only Download (8MB)

Abstract

Fusion-peptides (FP) in spike proteins (host attachment protein) are believed to be a key player that mediate early events in cell-to-cell fusion, which is vital for virulence and spread. A proline residue located at the central position of FP has often been suggested to have a distinctive role in cell-to-cell fusion facilitating viral entry and spread. We investigate the role of double and single central proline (s) in the FP from two very closely related recombinant strains of Mouse Hepatitis Virus (MHV; a β coronavirus), RSA59 and RSMHV2. While RSA59 (demyelinating strain) can readily cause cell-to-cell fusion, RSMHV2 (non-demyelinating strain) is fusion impaired. RSA59 (PP) contains two central consecutive proline(s) in the FP, when one proline is deleted, RSA59 (P) produces significant differences in neural cell syncytia formation and viral titer post infection in vitro. Comparatively, RSMHV2 (P) contains one proline in the FP, when extra proline is inserted, RSMHV2 (PP) produces small neural cell syncytia formation. Structural elucidation of the 16-mer FP fragment from RSA59 (PP) using Nuclear Magnetic Resonance spectroscopy revealed formation of cis and trans isomeric prolyl peptide states in the ratio of 40:60 in methanol, in contrast to 100% trans conformation of its 24-mer aqueous counterpart. The cis isomer with a helix-turn-helix structure is more ordered than its trans counterpart with less secondary structure. Comparative molecular dynamics studies on the trimeric fusion domain and FP of single and double proline variants of RSA59 and RSMHV2 spike proteins, reveal the unique role of proline due to its atypical stereochemistry. Two consecutive prolines imparts steric rigidity to the FP segment which is enhanced by formation of hydrogen bonds in the immediate neighborhood. Taken together, the observations suggest a dual role for the central proline, wherein in aqueous environment it imparts conformational rigidity to the fusion apparatus, and facilitates the same by promoting a more ordered structure in the membranotropic environment through ready isomerization to a cis peptide form. RSA59 (PP) and RSA59 (P) infection studies in C57Bl/6 mice by transcranial inoculation yielded distinct pathogeneses at day 3 post-infection. Both cause similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produces widespread encephalitis that extends deeply into the brain parenchyma. In contrast, RSA59 (P) infection is limited with minimal extension of viral antigen from the meninges and the inoculating needle tract. By day 6 post-infection, both viruses are mostly cleared from the brain. Interestingly, deletion of proline, RSA59 (P) significantly reduces demyelination at chronic stage of the disease compared to RSA59 (PP). Comparatively, RSMHV2 (NDM) strain does show the altered hepatitis and virus spread to brain parenchyma day 6 post-infection. At day 6 post-infection, the proline mutated NDM strain, RSMHV2 (PP) is able to penetrate in the white matter of spinal cord which is unusual for the NDM strain, but it was not able to cause demyelination in spinal cord white matter at chronic stage. Our work provides a premise for understanding genomic determinant of virus entry in CNS parenchyma and the subsequent altered pathogenesis.

Actions (login required)

View Item