Complex mechanisms for the packaging of the UL16 tegument protein into herpes simplex virus

Abstract

The conserved UL16 tegument protein of herpes simplex virus exhibits dynamic capsid-binding properties with a release mechanism that is triggered during initial virus attachment events. In an effort to understand the capsid association and subsequent release of UL16, we sought to define the mechanism by which this protein is packaged into virions. The data presented here support a model for the addition of some UL16 to capsids prior to their arrival at the TGN. UL16 was found on capsids isolated from cells infected with viruses lacking UL36, UL37 or gE/gD, which are defective for budding and accumulate non-enveloped capsids in the cytoplasm. Additionally, membrane-flotation experiments showed that UL16 co-purified with cytoplasmic capsids that are not associated with membranes. Moreover, the amount of UL16 packaged into extracellular particles was severely reduced in the absence of two conserved binding partners, UL21 or UL11.

FIG. 1

Potential UL16 packaging mechanisms. Mature DNA containing C-capsids exit the nucleus of infected cells via a budding then fusion mechanism across the nuclear envelope. It is at this step during the assembly process that UL16 could be loaded onto capsids through its direct interaction with the membrane-bound UL11 protein that is present on the nuclear envelope (nuclear loading model). Following exit from the nucleus, non-enveloped capsids transverse the cytoplasm and provide another potential location for the incorporation of UL16 (capsid loading model), possibly through an interaction with the capsid-bound UL21 protein. And finally, cytoplasmic capsids reach TGN-derived membranes where the final budding event is orchestrated. It is here that capsids acquire additional tegument proteins and their full complement of glycoproteins. Since UL11 targets to these membranes, it could bring UL16 to this location for packaging into virus particles (TGN loading model).

FIG. 2

UL16 is associated with non-membrane-bound capsids. Cells were infected with (A) HSV or a (B) mutant that does not make capsids (K23Z) for 20 h and mechanically lysed in a hypotonic buffer. Lysates were then loaded on the bottom of a sucrose step gradients, and centrifuged for 18 h to separate membranes (top) from non-membrane bound proteins (bottom). Fractions (800 μl) were collected from the top of the gradient, proteins in the fractions were then TCA-precipitated, separated by SDS-PAGE in 10% gels and analyzed by immunoblot with antibodies specific for gE, VP5, and UL16. (C) The bottom two non-membrane associated fractions from the gradients were incubated in NP-40 lysis buffer for 15 min. Capsids in these fractions were subsequently pelleted through a 30% (wt/vol) sucrose cushion and analyzed by immunoblot.

FIG. 3

UL16 is bound to cytoplasmic capsids that are blocked in maturation budding. Cytoplasmic capsids from detergent lysed (A) WT, ΔUL37, or gE-/gD- infected cells were sedimented in 20 to 50% (wt/vol) sucrose gradients for 1 h. Fractions containing capsid bands were pulled with a needle syringe and pelleted through another 30% sucrose cushion. Purified capsids and associated proteins were separated by SDS-PAGE in 10% gels and analyzed by immunoblot with anti-VP5, anti-UL37, anti-UL16, anti-gE, and anti-gD rabbit serum. (B) ΔUL36 and ΔUL37 capsids were isolated from infected cells in parallel with WT as described above. cyto, cytoplasmic. WT, wild-type.

FIG. 4

UL16 is packaged and associates with cytoplasmic capsids in the absence of UL21. Cytoplasmic capsids from detergent lysed HSV or ΔUL21 infected cells were sedimented in a 20 to 50% (wt/vol) sucrose gradient for 1 h. Regions of the gradient containing capsid bands were isolated with a syringe and then pelleted through a 30% (wt/vol) sucrose cushion. Virions present in the media were sucrose gradient purified as described. Virions and capsids with their associated proteins were separated by SDS-PAGE in 10% gels and analyzed by immunoblot with VP5 and UL16 specific antibodies. cyto, cytoplasmic.

FIG. 5

An interaction with UL11 is not essential for UL16 packaging. WT or ΔUL11 extracellular virus and cytoplasmic capsids were purified in 20-50% (wt/vol) sucrose gradients and the region of the gradient containing the virions and capsids were pulled with a needle syringe and pelleted through a sucrose cushion. Cell lysates, purified capsids, and virions were then analyzed for the presence of VP5, UL16, and UL11 by immunoblot. cyto, cytoplasmic.