Abstract
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498 HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.
Highlights
Human cytomegalovirus (HCMV) morphogenesis has been studied extensively by transmission electron microscopy (TEM) [1,2,3,4,5,6,7]
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV)
Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which may be used by nucleocapsids for budding
Summary
Human cytomegalovirus (HCMV) morphogenesis has been studied extensively by transmission electron microscopy (TEM) [1,2,3,4,5,6,7]. As a member of the betaherpesvirinae subfamily, HCMV particles consist of (I) the capsid, a stable protein shell that harbors the viral genome; (II) the tegument, a proteinaceous layer associated with the capsid; and (III) the virus envelope, a lipid bilayer originating from the host cell, which surrounds the tegumented capsid. The enlarged host cell nucleus is the site of capsid formation as well as DNA synthesis and encapsidation. In some cases the scaffold is degraded and DNA encapsidation does not take place which leads to the formation of A capsids [11,12,13,14]. The nuclear stage ends with the transition of tegumented capsids from the nucleus into the cytoplasm where final virus tegumentation and envelopment occurs in the viral assembly complex
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