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The CVSC mechanically reinforces HSV-1

ABSTRACT: Using atomic force microscopy imaging and nanoindentation measurements, the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of the icosahedral Herpes Simplex Virus type 1 capsids was investigated. pUL17 and pUL25 that form the capsid vertex-specific component particularly contributed to the capsid resilience along the 5-fold and 2-fold, but not the 3-fold icosahedral axes. We tested whether deletion of UL17 and UL25 changed the levels of the proteins that assemble in the nucleocapsid by quantitative mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Herpes Simplex Virus (type 1 / Strain F)

SUBMITTER: Eleonora Corradini

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD005104 | Pride | 2017-04-20

REPOSITORIES: Pride

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			Action	DRS
	OR10_20151030_EC_HHV1_AUL25.msf	Msf
	OR10_20151030_EC_HHV1_AUL25.raw	Raw
	OR10_20151030_EC_HHV1_AUL25_02.msf	Msf
	OR10_20151030_EC_HHV1_AUL25_02.raw	Raw
	OR10_20151030_EC_HHV1_A_02.msf	Msf

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Project description:Rhinovirus causes the common cold and drives exacerbation in asthma and COPD, leading to substantial morbidity and healthcare cost. The single positive strand RNA genome of this non-enveloped virus is translated into a single polyprotein that is processed by virus 3C protease to form three structural capsid proteins VP0, VP1 and VP3, and other proteins required for viral life cycle completion. Capsid proteins assemble into the 5S capsid protomer, five of which form a 14S pentamer, and 12 pentamers then assemble to form the 150S icosahedral provirion into which the viral genome is incorporated. During the final step of viral maturation, VP0 is cleaved into VP2 and VP4. VP4 is encoded at the N-terminus of the viral polyprotein, and in many Picornaviruses is N-myristoylated by host cell N-myristoyltransferase (NMT). We have explored the impact of IMP-1088 (the first sub-nanomolar IC50 dual inhibitor of human N-myristoyl transferases HsNMT1 and HsNMT2) on HRV capsid myristoylation in cells. Global identification of proteins for which myristoylation is selectively inhibited by IMP-1088 was performed by quantitative chemical proteomic analysis of YnMyr-tagged proteins in HeLa cells infected with RV16 treated with 50 nM IMP-1088 or vehicle (DMSO). Proteins were subjected to ligation to AzRB, an azide capture reagent bearing a trypsin-cleavable linker and biotin dual label followed by affinity enrichment on Neutravidin beads, on-bead digestion with trypsin, nanoLC-MS/MS analysis of tryptic peptides, and data processing by label-free protein quantification (LFQ) in MaxQuant. 60 N-myristoylated proteins were identified, including the RV16 polyprotein which responded to IMP-1088 inhibition, with no significant change in protein abundances across the whole proteome in the presence of inhibitor. The majority of viral capsid peptides identified in the pull-down mapped to the VP0 domain of the polyprotein, and the N-terminal YnMyr-tagged peptide was readily identified by MS/MS. These data provide the first direct evidence for HRV capsid myristoylation in human cells, and demonstrate that this lipid modification is highly susceptible to inhibition by a selective NMT inhibitor.

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The CVSC mechanically reinforces HSV-1

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