RESUMEN
Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways. We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity.
Asunto(s)
Citomegalovirus , Ensamble de Virus , Citoplasma/metabolismo , Humanos , ViriónRESUMEN
The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.
Asunto(s)
Proteínas E3 de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Membrana Nuclear/metabolismo , Proteínas E3 de Adenovirus/genética , Línea Celular Tumoral , Humanos , Lamina Tipo A/metabolismo , Microscopía Electrónica de Rastreo , PermeabilidadRESUMEN
The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.