Tegumentation and Envelopment

The composition of the HCMV tegument and envelope, and the relationship of their acquisition to virus egress from the nucleus and cell, have recently been reviewed (Eickmann et al. 2006). The cartoon shown in Fig. 7 serves to summarize some of the steps involved. A general consensus of data supports an envelopment/ de-envelopment mechanism for nuclear egress of herpesviruses, followed by final envelopment through cytoplasmic membranes (Severi et al. 1988; Gibson 1993; Enquist et al. 1998; Mettenleiter 2002; Leuzinger et al. 2005; Campadelli-Fiume and Roizman 2006). As represented in Fig. 7, step 1, primary envelopment of the capsid occurs at the inner nuclear membrane, enabling the particle to pass into the perinuclear space.

Primary envelopment requires two herpesvirus group-conserved proteins. Their HCMV homologs are pUL50 and pUL53; the respective HSV homologs are pUL34 and pUL31 (Klupp et al. 2000; Roller et al. 2000; Reynolds et al. 2001; Fuchs et al. 2002; Reynolds et al. 2002). HSV pUL34 localizes to both the inner and outer leaflets of the nuclear membrane and is a substrate for the virion-associated US3 protein kinase (Purves et al. 1992), which enhances its membrane localization (Purves et al. 1992; Klupp et al. 2000, 2001; Roller et al. 2000; Reynolds et al. 2001, 2002). Although CMV does not encode a US3 homolog, the murine CMV homolog of pUL34 (MCMV M50/p35) interacts with cellular protein kinase C and carries it to the nuclear membrane where it is proposed to phosphorylate the

Fig. 7 Egress of nucleocapsid. Shown here is a drawing illustrating some features of the herpesvirus egress pathway. 1 Intranuclear capsids bud through the inner nuclear membrane by a primary envelopment process requiring the homologs of CMV pUL50 and pUL53 (represented by clustered short lines on inner and outer nuclear membranes). 2 Resulting enveloped particle in perinuclear space represents a translocation intermediate. 3 De-envelopment at the outer nuclear membrane releases the nucleocapsid into the cytoplasm where it acquires final complement of tegument proteins (small lines and circles; also depicted in nucleus and perinuclear space to indicate uncertainty about site(s) of addition). 4 Fully tegumented capsid buds into cytoplasmic vesicles or tubules, through which it completes egress from the cell. The presence of tegumented B-capsids undergoing secondary envelopment in the cytoplasm (see Fig. 1d), indicates this mechanism is not selective for DNA-containing particles

Fig. 7 Egress of nucleocapsid. Shown here is a drawing illustrating some features of the herpesvirus egress pathway. 1 Intranuclear capsids bud through the inner nuclear membrane by a primary envelopment process requiring the homologs of CMV pUL50 and pUL53 (represented by clustered short lines on inner and outer nuclear membranes). 2 Resulting enveloped particle in perinuclear space represents a translocation intermediate. 3 De-envelopment at the outer nuclear membrane releases the nucleocapsid into the cytoplasm where it acquires final complement of tegument proteins (small lines and circles; also depicted in nucleus and perinuclear space to indicate uncertainty about site(s) of addition). 4 Fully tegumented capsid buds into cytoplasmic vesicles or tubules, through which it completes egress from the cell. The presence of tegumented B-capsids undergoing secondary envelopment in the cytoplasm (see Fig. 1d), indicates this mechanism is not selective for DNA-containing particles nuclear lamina proteins underlying the inner nuclear membrane, weakening their interaction and dissolving the barrier they pose to capsid egress (Muranyi et al. 2002). Interaction of the membrane-associated member of the nuclear-egress pair (e.g., CMV pUL50) with its nuclear phosphoprotein partner (e.g., CMV pUL53) may then drive primary envelopment (Mettenleiter 2002; Muranyi et al. 2002; Bjerke et al. 2003).

Immature particles in the perinuclear space are then proposed to bud through the outer leaflet of the nuclear membrane, loosing their translocation membrane and becoming nonenveloped cytoplasmic particles (Fig. 7, step 2). Although this general pathway appears to be shared by all herpes viruses, there is less consensus about where the tegument proteins are added.

With regard to the predominant tegument proteins of CMV, three sets of observations are compatible with their proposed addition outside the nucleus (Fig. 7, step 3). First, electron microscopy shows that cytoplasmic capsids have a thick fibrillar coating (deduced to be tegument proteins) that is entirely absent from nuclear capsids (Figs. 1C, 1D, 6; Fig. 1 in Gibson 1993). Second, SDS-PAGE analyses show that capsids recovered from the cytoplasm of infected cells contain the predominant tegument proteins, whereas those from the nucleus do not (Gibson 1981). And third, immunofluorescence studies show accumulations of three abundant tegument proteins (pUL83, pUL32, pUL99) in assembly compartments juxtaposed to the nucleus, but outside of it (Scholl et al. 1988; Sanchez et al. 2000a, 2000b) (reviewed in Eickmann et al. 2006). None of these observations, however, rule out the possibility that some or all of the same tegument proteins bind to capsids within the nucleus or perinuclear space (Hensel et al. 1995; Nii et al. 1998) and are rapidly translocated with the capsid into the cytoplasm where they accumulate to levels more readily detected. Compatible with this possibility, recent studies show that specific mutations in the tegument proteins pUL36 (VP1/2) of HSV, or pUL32 (basic phosphoprotein/pp150) of HCMV, result in their accumulation within the nucleus (O'Hare and Abaitua 2006; J. Wang and W. Gibson, unpublished data from studies using mutant viruses encoding CysCysProGlyCysCys-tagged pUL32 detected in live, infected cells with the biarsenical dye FIAsH).

Once in the cytoplasm and completely tegumented, the capsids bud into cyto-plasmic vesicles or tubules to acquire their final envelope (e.g., Fig. 7, step 4). This process required pUL99; in its absence, tegumented capsids accumulate in the cytoplasm (Silva et al., 2003). Several changes appear to accompany this process; including compression or tightening of the tegument and thickening of the membrane with apparent elaboration on its luminal surface. These changes occur where the membrane and capsid are in close proximity (e.g., Fig. 1d), suggesting conformational or compositional changes in both layers as envelopment proceeds. Late maturational events such as phosphorylation by the virion-associated kinase(s) (Roby and Gibson 1986; Nogalski et al. 2007), carbohydrate processing, and possible redistribution of envelope and tegument constituents, are likely to occur as the particle completes its egress and is modified to increase its efficiency as an entry vessel for delivering the viral DNA to the next cell.

The recently discovered ubiquitin-specific cysteine protease (DUB) activity, present and functional in virions at the amino end of the high-molecular-weight tegument protein pUL48 of HCMV and pUL36 of HSV (Kattenhorn et al. 2005; Schlieker et al. 2005; Wang et al. 2006), may be involved in these late events, or have a role at the outset of infection, or both. Viruses mutated in the catalytically critical Cys41 or His163 residues of the HCMV DUB replicate at a reduced efficiency relative to wild type virus, indicating an important even if not absolutely essential role for this new virion-associated enzyme (Wang et al. 2006).

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