Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

Abstract

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsidmembrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCEMembrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment. Herpesviruses replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is then generally accepted that capsids bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These naked cytoplasmic capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (4-9). Completion of cytoplasmic envelopment requires the coordinated interaction of multiple virally encoded proteins (8,(10)(11)(12)(13) and the participation of components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery (14-19).UL36p (VP1/2) is the largest structural protein encoded by the herpesviridae (20, 21). It is a major constituent of the innermost layer of tegument, the complex protein layer between the capsid and the inner surface of the envelope, and connects the capsid to m...

Herpes Simplex Virus Capsid Localization to ESCRT-VPS4 Complexes in the Presence and Absence of the Large Tegument Protein UL36p

“…We prepared a postnuclear supernatant (PNS; cytoplasmic fraction) from GS2822-or GS3494-infected HEK293 cells expressing the dominant negative Vps4-EQ allele fused to GFP. The PNS was attached to polylysinecoated MatTek dishes, fixed, and imaged by fluorescence microscopy in the red (VP26-mRFP1 capsid subunit) or green (Vps4-EQ-GFP) channel, as we described earlier (48). Typical fluorescence fields and galleries of individual particles are shown for the parental strain GS2822 (Fig.…”

“…We showed earlier, by using infected-cell extracts and organelles prepared in vitro, that HSV capsids dock to cytoplasmic organelles in the absence of UL36p (48). To test whether capsid/membrane docking occurred in vivo in intact cells in the absence of UL36p, we infected Vero or UL36p-expressing HS30 cells with the UL36-null GS3494 strain and then fixed and sectioned cells for transmission electron microscopy (Fig.…”

“…Direct fluorescence microscopy studies were performed as previously described (48,62) in the Analytical Imaging Facility of the Albert Einstein College of Medicine. PNS samples were attached to 35-mm glass-bottomed MatTek dishes (MatTek Corporation) precoated with 100 g/ml poly-L-lysine (Sigma).…”

“…One important function of UL36p is to recruit UL37p (19,20,25,29,44,45), a putative mimic of cellular multisubunit tethering complexes (28) that mediates capsid docking to organelles, including the trans-Golgi network (TGN) (27), perhaps via membrane anchors, such as the heterodimer gK/UL20p (46). In the absence of UL36p/UL37p, capsid/membrane docking can still occur, but the fidelity of capsid-organelle targeting appears to be impaired (47,48). Stable and precise capsid/membrane association prior to envelopment may require cooperation between UL36p/UL37p and complexes containing the UL16p protein (21,39,49,50).…”

ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells

“…tegument proteins US3, UL47, or UL49, cellular F-actin is incorporated into the recombinant PRV particles to produce mature virions (44,45). HSV-1 pUL37 recruits pUL36 (41,46,47) and interacts with glycoprotein K and membrane protein UL20 for efficient cytoplasmic envelopment (48). Intact virions were produced in 7D-infected ARPE-19 cells and NPCs, but not in the differentiated neuronal cells of dNPCs and dSY5Y.…”