Open reading frame number 16 within the unique-long segment of the herpes simplex virus (HSV) genome encodes a 373-amino-acid (aa) protein whose function is almost entirely unknown, as is the case for the homologs found in all families (alpha, beta, and gamma) of herpesviruses (25
). The UL16 protein is expressed late in the infection and initially accumulates in the nucleus, but at later times is found primarily in the cytoplasm (48
). When virions bud into cytoplasmic membranes, UL16 is packaged into the tegument—the layer of the virion situated between the capsid and the viral envelope (50
). Mutants that do not express UL16 are viable but produce only ~10% the number of infectious virions compared to the wild type in cell cultures (3
). Thus, this protein plays an augmenting role in the replication cycle; one that is highly conserved.
Previous studies have suggested two potential functions for UL16. First, it may provide one of the bridging functions that link capsids to membranes during the envelopment process within the cytoplasm. In support of this hypothesis, a population of UL16 molecules has been found that is associated with cytoplasmic capsids (48
), and there is a strong interaction between UL16 and UL11 (43
), a small tegument protein that is peripherally bound to membranes via two covalently attached fatty acids, myristate and palmitate (6
). Like UL16, UL11 is needed for efficient envelopment and is conserved among all of the herpesviruses (4
The second potential function for UL16 comes from studies of extracellular virions. These showed that binding of the virus to attachment receptors (heparan sulfate), either on the surface of host cells or immobilized on agarose beads, causes a signal to be sent into the tegument to trigger the release UL16 from the capsid (49
). The purpose of this rearrangement in the tegument is unknown, but it could be important for uncoating of the capsid and/or activation of the fusion apparatus prior to virus entry. In any case, it is clear from studies of UL16 that the assembly of the tegument creates machinery with moving parts that respond to signals detected on the outside of the virion.
To understand how the tegument machine is assembled and activated, a thorough understanding is needed of the network of interactions in which UL16 operates. Prior to the experiments described here, three interactions were known. One is the interaction with UL11, and within that protein, UL16 specifically recognizes a cluster of acidic residues (43
). Attempts to map the part of UL16 involved in this interaction were not successful, but modification of its free cysteines with N
-ethylmaleimide (NEM) blocked binding to UL11 (81
). A second protein with which UL16 interacts is UL21, a tegument protein that is conserved among alphaherpesviruses and is also bound to capsids (2
). This interaction involves a domain in the second half of UL21 (28
), but again, the part of UL16 that participates in binding remains unknown. The site must be distinct from that used for UL11 because binding to UL21 is not blocked with NEM (28
). Evidence for a third protein in the interaction network comes from the discovery that UL21 is not required for binding of UL16 to capsids (47
). The unidentified protein must be a part of the capsid structure or another tegument protein that is bound to capsids. Although the UL16-capsid interaction is destabilized upon binding of the virion to its attachment receptors (49
), other changes in the network remain to be elucidated.
To receive the signal from outside the virion, the UL16-interaction network must interface in some manner with viral glycoproteins on the surface. The only clear linkage to the membrane is via UL11; however, because that protein does not span the membrane, another network connection with a viral glycoprotein must exist. There is one report of a possible interaction between UL11 and the tails of glycoprotein D (gD) and gE (22
), but the data are limited and have been suggested to be the result of nonspecific binding (32
). The companion paper (26a
) shows that the gE-UL11 interaction indeed occurs and is biologically significant; however, neither gE nor gD binds to attachment receptors. That function is provided by gB and gC (10
). There is some evidence that gB and gC reside in a complex with gD and gE on the surface of the virion (27
), and thus signals might be sent between glycoproteins prior to being routed into the tegument. Clearly, a much better understanding of tegument-glycoprotein “wiring” is needed.
The experiments described below began with the question of whether UL16 interacts directly with the cytoplasmic tail of gB, one of the glycoproteins known to bind attachment receptors (66
). The data quickly ruled against that hypothesis. Instead, converging lines of evidence suggested that UL16 interacts with gE, a glycoprotein that has long been known to be required for the spread of HSV in a cell-to-cell manner (33
). Rigorous proof that the interaction can occur within cells required the fortuitous discovery of a particular UL16 mutant, one that also provides evidence that the interaction with gE is regulated.