Encapsidation of the newly replicated genome is an important step in the maturation and assembly of all viruses. The fundamental aspects of this macromolecular assembly process have been conserved in organisms ranging from bacteriophages to viruses infecting mammalian cells. Assembly involves interactions between the preformed capsids, the viral nucleic acid, and the packaging machinery. The packaging machinery is believed to generate the required forces and direct the genome encapsidation. The processes of genome maturation and cleavage and packaging have been studied extensively with model systems including bacteriophage lambda (4
), Φ29 (6
), and P22 (27
), which serve as prototypes for the study of the complex molecular mechanisms in viruses which infect eukaryotes.
By analogy with the better-studied bacteriophages, the HSV-1 portal protein has an important role in the encapsidation process, serving both as a channel for DNA insertion and as a docking site for the other components of the packing machinery (17
). The experiments described in this paper were designed to map regions of the UL6 protein that mediate its interaction within the ring and with the other cleavage and packaging proteins. Nine of the ten deletion mutants failed to complement the null virus despite detectable levels of protein expression. The portal protein is involved in interactions with multiple partners during cleavage and packaging of the HSV-1 genome, and the deletions may thus affect one or more of these interaction domains, rendering the UL6 gene relatively intolerant to mutations. The high degree of sequence similarity seen in the portal proteins from different herpesviruses is also consistent with this interpretation. We cannot rule out, however, that some deletions may have caused conformational changes in the UL6 protein. The complementing RGD mutant contains a seven-amino-acid deletion eliminating a putative integrin binding motif, indicating that this motif is not essential for UL6 function. A second RGD sequence (238 to 240) is also present within the UL6 protein; this motif is located within the sequences deleted in the D-5 mutant. In the phage Φ29 terminal protein, the RGD motif is required for its interaction with the viral DNA polymerase (11
). It is possible that the second RGD motif in UL6 (deleted in the D-5 mutant) is involved in protein-protein interactions.
Three deletion mutants, D-5, D-6, and D-LZ, and a double point mutant, the L429E L436E mutant, were introduced into the viral genome for phenotypic analysis, and all four were shown to produce only immature B capsids. This is consistent with the phenotype of the previously described UL6 null mutant and of mutants in other components of the cleavage/packaging machinery. Most mutations isolated to date in the UL6, UL15, and UL28 genes show this “all or none” phenotype. It has been hypothesized that DNA packaging and capsid maturation are coupled processes, hence the difficulty in separating these events.
The D-5 protein was localized to the cytoplasm of infected cells and was not incorporated into capsids; furthermore, it could not be expressed stably in insect cells from recombinant baculoviruses. Interestingly, Preston and colleagues reported a UL6 insertion mutant (UL6 in269) in which the UL6 protein failed to localize to the nucleus; moreover, this mutant UL6 protein also appeared to retain both UL15 and UL28 in the cytoplasm (30
). The UL6 amino acid sequence does not reveal any of the canonical nuclear localization sequence motifs; however, since the deletion of amino acids 198 to 295 in the D-5 mutant and an insertion at residue 269 prevented nuclear import, it is tempting to speculate that this region may contain an unrecognized NLS or domain of interaction with another NLS-containing protein. The ability of the UL6 in269 mutant to retain UL15 and UL28 in the cytoplasm suggests that the region of UL6 required for interaction with UL15 and UL28 does not reside in the region disrupted by this insertion. Taken together, these results suggest that the D-5 mutant may lack an NLS, but we cannot rule out that this protein is mislocalized due to a folding defect which may also contribute to its instability. Whether the D-5 protein can still interact with the terminase is not clear because of difficulties expressing this protein in insect cells.
The deletion mutants D-6 and D-LZ and the L429E L436E double point mutant are able to enter the nucleus, localize to the replication compartments, and are incorporated into B capsids. UL15 can still associate specifically with B capsids formed in cells infected with these mutants (data not shown); however, these mutants exhibit a severe defect in cleavage and packaging. The D-6 protein, however, was found to be competent to form oligomeric ring structures in vitro. This result suggests that this mutant is deficient in DNA packaging, perhaps due to changes in the properties of the channel which controls the entry and/or exit of DNA into or out of the capsid (12
). Within the deleted region of D-6, sequence analysis of the UL6 protein predicts a potential alpha-helix and a sequence similar to the DNA binding region of integrases. The DNA channels of portal proteins for which atomic structures are known contain alpha-helices and are known to interact with the DNA. The D-6 mutant could be defective in these interactions, which may explain its inability to package DNA. It will be necessary to introduce point mutations within this region and test them for DNA interactions to address this possibility. Another possible reason for the defective DNA packaging of this mutant may be due to the loss of interaction with other components of the packaging machinery. The UL15 protein has been shown to be transiently associated with procapsids (22
) and with B capsids (21
). The observation that UL15 does not specifically associate with capsids isolated from cells infected with the UL6 null mutant virus implies an interaction between UL15 and UL6 (33
). Using immunoprecipitation assays, a direct interaction between UL6 and UL15 and UL28 was also observed in recombinant baculovirus-infected insect cells (30
). However, we did not see any difference in levels of UL15 in the capsids isolated from the D-6 mutant virus, indicating that terminase association is not affected in this mutant (data not shown). Thus, this mutant may be reminiscent of DNA channel mutants described for the Salmonella
phage SPP1 portal (12
The results described in this article indicate that the leucine zipper in UL6 is required for assembly of portal-containing HSV-1 capsids which are competent for encapsidation. There is precedent for the use of leucine zippers in proteins that form rings. For example, the leucine zipper in phospholamban (a protein which regulates the levels of intracellular calcium) appears to play an important role in stabilizing the pentamers of this protein to form a coiled-coiled pore (23
). Based on our finding that the D-LZ and L429E L436E mutants fail to form rings in vitro, we suggest that this region is important for the formation and/or stability of the HSV-1 portal ring. Interestingly, despite the inability of the recombinant proteins to form rings in vitro, the D-LZ and L429E L436E mutant proteins were associated with B capsids from infected cells. The association with capsids may indicate that the interaction domain between mutant UL6 and capsid proteins is not affected. The amount of UL6 present in mutant capsids was less than that observed in WT capsids, suggesting that the D-LZ and L429E L436E mutant proteins are not efficiently incorporated into capsids during assembly. It is possible that the leucine zipper mutant proteins form polymorphic aggregates that can initiate assembly, resulting in capsids with a lower ratio of UL6 to VP5. Aggregate formation might be expected if additional protein-protein interactions contribute to the formation of rings. The forces which stabilize UL6 rings are not known but could include electrostatic or hydrophobic interactions, disulfide linkages, and/or other structural motifs. In fact, we have preliminary data showing that intersubunit disulfide bonds involving two cysteines of the UL6 protein are also important in portal ring formation (J. Nellissery and S. Weller, unpublished data). Intersubunit disulfide bond formation may result in the formation of aggregates which still associate with capsids but are defective for encapsidation. This aggregation may explain the sedimentation behavior of the D-LZ and L429E L436E mutant proteins and the fact that monomers are not observed.
Recently the three-dimensional structure of the bacteriophage SPP1 portal protein was determined at 3.4 Å (14
). Each subunit consists of a crown, wing, stem, and clip domains. Twelve to thirteen of these monomers are arranged in a ring around a central channel, similar to the portals of other bacteriophages and herpesviruses. The channel is lined by loops which protrude inward and are positioned so as to contact the major groove of the DNA; this model suggests that the movement of these loops is coupled to DNA translocation. The portal proteins of bacteriophages and herpesviruses show little sequence similarity, but their secondary structures display significant conservation of helices, sheets, and loop regions. When we aligned the predicted secondary structure of the UL6 protein to that of the SPP1 portal protein, we observed that our deletion mutants mapped to the wing (D-5), stem and channel (D-6), and major helix α6 (D-LZ and L429E L436E) domains. The D-6 mutant is particularly interesting since it lacks the channel loops that are thought to play a dynamic role during DNA packaging. The leucine zipper mutants (D-LZ and L429E L436E mutants) map to the conserved helix α6; the leucine zipper which is present within this region in alphaherpesviruses may be important in interactions that affect the stability of the portal ring.
In summary, we have generated four HSV-1 portal protein mutants that are defective in cleavage and packaging of the genome and have determined the localization of their gene products within infected cells and within viral capsids. We have also shown that the WT and one mutant (D-6) protein formed oligomeric rings in vitro, while the two other mutants (D-LZ and L429E L436E mutants), in which the putative leucine zipper is disrupted, did not form stable rings. This is the first report defining a region within the UL6 protein that is essential for the formation of stable portal rings.