Taking control: Human cytomegalovirus invades host cellular organelles for virion assembly, maturation, and release

摘要

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that infects over 60% of adult population. Whilst HCMV infections are generally asymptomatic in healthy individuals, it is a significant cause of morbitiy and mortality in immuno-compromised people, and a major cause of birth defects. The HCMV virion comprises a nucleocapsid that houses the double-stranded DNA genome, and is surrounded by a proteinaceous tegument layer, and glycoprotein-containing lipid envelope. Capsid assembly and packaging of viral DNA precedes nuclear egress, and is followed by cytoplasmic tegumentation and secondary envelopment before extracellular release (Figure 1). However, precise temporal and spatial acquisition of the tegument and viral membrane, and mechanisms for release of the infectious virion that can be targeted for therapeutic intervention remain largely undefined. To determine which host sites are hijacked for virion assembly and maturation, organelle isolation and fractionation was optimized in lung fibroblast MRC5 cells. Using known organelle markers, a robust and reproducible technique employing density-based organelle separation was established. This was followed by collection of individual organelle fractions from each uninfected (MOCK) and HCMV infected (strain AD169) MRC5 cells at 24, 48, 72, and 96 hours post infection (hpi). Individual organelles were lysed, resolved by SDS-PAGE, and analyzed in 186 individual Nlc-MS/MS runs per biological replicate.