Investigating the spatio-temporal organization of human cytomegalovirus secondary envelopment and egress

Abstract

The human cytomegalovirus (HCMV) is an opportunistic pathogen of high clinical importance. This virus can cause severe disease and disabilities, especially in vulnerable patient groups with a weakened or immature immune system. Despite continuous efforts and more than 60 years of HCMV research, no vaccine has been approved to date. Inhibitors of viral replication approved today for clinical use exclusively target nuclear stages of HCMV replication, whereas no approved pharmaceuticals target the later cytoplasmic steps of viral morphogenesis. An HCMV particle acquires its final envelope in a step called secondary envelopment before egressing the cell. This process is critical for the virus since the final envelope must contain all factors necessary to mediate entry into the next host cell. To achieve this, HCMV significantly remodels cell membranes and trafficking processes to form a specialized viral assembly complex (AC).

The models of herpesvirus secondary envelopment currently prevailing in the literature suggest that single capsids are individually enveloped by budding into single vesicles. These double enveloped particles can subsequently be released by fusion with the plasma membrane. This study focused on the spatio-temporal organization of HCMV secondary envelopment and egress, examining the dynamics and mechanics of the release of viral progeny. Experiments utilizing high-end live-cell microscopy such as lattice-light-sheet- and total internal reflection fluorescence (TIRF) microscopy revealed that, in contrast to the models mentioned above, large amounts of HCMV viral material are exocytosed in intermittent pulses from large bodies in infected cells. Correlative light and electron microscopy investigations showed that these bodies are multivesicular structures filled with virus particles and dense bodies (viral tegument particles without capsid and genome). Experiments with pH-sensitive reporter proteins show that these multiviral bodies (MViBs) release their content to the extracellular medium by fusion with the plasma membrane. These release events produce characteristical extracellular viral accumulations (EVAs) at the surface of infected cells. Moreover, an analysis of the composition of virions and MViBs confirms the presence of endosomal and exosomal markers such as CD63 on MViBs as well as e.g. Rab5C and Syntaxin-12 on virions. Together with bulk release phenotype, these data suggest the involvement of endosomal and exosomal pathways in the MViB generation. However, experiments with inhibitors of classical MVB biogenesis (U18666A) and exosome release processes (Ketotifen and Tipifarnib) showed that the viral process that generates MViBs likely differs considerably from the cellular process to produce MVBs. Only Tipifarnib could interfere with viral replication, whereas U18666A and Ketotifen had no significant effect. Interestingly, the prevalence of the release from MViBs into EVAs varied between the HCMV strains TB40 and Merlin, suggesting a role for bulk release in strain-specific spreading behaviour.