To map the domains of portal protein pUL6 that are critical for interaction with scaffold proteins, UL6 was cloned in frame with a Flag epitope. Full-length and truncated versions of this plasmid were transfected into CV1 cells with a pUL26 expression plasmid. Lysates of the transfected cells were then subjected to immunoprecipitation with scaffold-specific antibody, and the presence or absence of scaffold and portal proteins in electrophoretically separated immunoprecipitated material was determined by immunoblotting.
As shown in Fig. , the anti-scaffold antibody immunoprecipitated VP22a, the major scaffold protein, in all cases and pUL26 in most cases. Moreover, Flag-pUL6 was expressed in contransfected cells to readily detectable levels, as revealed on immunoblots of the cellular lysates probed with M2 antibody. Most importantly for the purposes of this experiment, full-length pUL6 was coimmunoprecipitated with anti-scaffold antibody whether the Flag epitope was located at the N or the C terminus. Examination of truncated pUL6 revealed that amino acids 1 to 18 and 641 to 676 were dispensable for coimmunoprecipitation with scaffold proteins, whereas more extreme truncations than these precluded the interaction with scaffold proteins. We concluded that amino acids 19 to 640 of pUL6 were sufficient to interact with scaffold proteins as determined by immunoprecipitation.
FIG. 2. Mapping regions of pUL6 required for scaffold protein interactions. CV1 cells were cotransfected with expression plasmids encoding full-length pUL26 and Flag-tagged pUL6 or its derivatives. (A) Twenty-four hours after transfection, cells were harvested (more ...)
We next focused on residues within the identified scaffold interaction region of pUL6 that might be critical to its interaction with scaffold proteins. Given our previous results showing that a hydrophobic domain in scaffold proteins mediated interaction with the portal protein, we reasoned that hydrophobic residues within the portal might be important. We noted several highly conserved tryptophan residues within the portal protein (see Fig. ). To test whether these amino acids were important for scaffold protein interaction, we mutated each tryptophan codon to alanine and tested each mutant protein for its ability to interact with the scaffold protein by coimmunoprecipitation.
FIG. 7. Sequence alignments showing that tryptophan residues are conserved in the portal proteins of alphaherpesvirus. BHV, bovine herpesvirus; CHV, cercopithecine herpesvirus; EHV, equine herpesvirus; GaHV, gallid herpesvirus; HSV, herpes simplex virus; MeHV-1, (more ...)
As shown in Fig. , mutation of tryptophan residues at position 27, 90, 127, 163, 241, 262, 532, or 596 completely precluded coimmunoprecipitation by scaffold-specific antibody despite ample levels of the mutated portal proteins in the lysates. We concluded that these residues are critical to interaction with the scaffold protein in the absence of other viral proteins.
FIG. 3. Replacement of tryptophan by alanine within pUL6 abolishes its interaction with scaffold protein in transfected cells. CV1 cells were cotransfected with expression plasmids bearing full-length UL26 and wild-type UL6 or UL6 genes containing substitutions (more ...)
To test for the effects of the W/A mutations on viral replication, CV1 cells were transfected with plasmids bearing vector DNA, wild-type pUL6, or pUL6 bearing the W/A mutations at position 27, 90, 127, 163, 241, 262, 532, or 596. Expression of mutant and wild-type pUL6 was verified by immunoblotting 24 h later (not shown), and the pUL6-expressing cells were infected with a UL6 null virus. Twenty-four hours after virus infection, the cells were lysed, and the yield of infectious virus was determined on a UL6-expressing cell line, CV6. As shown in Table , expression of wild-type pUL6 rescued replication of the UL6 null virus, producing a final infectious titer of approximately 3 × 105 PFU/ml. In contrast, W/A mutations at positions 27, 90, 127, 241, 262, and 532 completely precluded rescue of viral infectivity, indicating that these residues were critical to portal function. Compared to wild-type pUL6, the W163A-bearing plasmid was reduced by approximately 1,000-fold in its ability to rescue replication of the UL6 null mutant, producing an infectious titer of approximately 300 PFU/ml. In contrast, the plasmid bearing W596A rescued viral replication as well as the wild-type plasmid, despite the observation that the mutation precluded coimmunoprecipitation of pUL6 and scaffold proteins when these proteins were coexpressed in the absence of virus infection (Fig. ). We conclude that other functions in virus-infected cells can compensate for the diminished interaction between the portal and the scaffold in cells expressing pUL6 W596A.
To investigate this idea further, recombinant viruses bearing (i) the W596A mutation and (ii) W27A (as an example of the group of mutations precluding replication in our complementation assay) were constructed using BAC technology and were designated vJB31(W596A) and vJB30(W27A), respectively. A virus derived from vJB30 but bearing a restored UL6 gene was also constructed and designated vJB30R.
As shown in Table , vJB31(W596A) replicated to levels similar to those of the wild-type virus in both CV1 cells and CV6 cells, which are derived from CV1 cells but express wild-type pUL
). In contrast to these results, neither vJB30(W27A) nor the UL
6 null mutant replicated to an appreciable extent on CV1 cells. vJB30(W27A) was able to propagate on UL
6-complementing cells to levels similar to that of wild-type virus, indicating that UL
6 was solely responsible for the replication defect in CV1 cells. This conclusion was further confirmed by the observation that vJB30R was able to replicate normally on CV1 cells.
To determine the fate of the portal-scaffold interaction in cells infected with vJB30(W27A) and vJB31(W596A), cells were infected with these viruses or HSV-1(F), and the amount of pUL
6 in clarified or total lysates was determined by immunoblotting. As shown in Fig. , steady-state levels of pUL
6 accumulated to similar levels in cells infected with JB30, JB31, and HSV-1(F) (Fig. ). On the other hand, slightly reduced levels of pUL
6 were present in clarified lysates of vJB30(W27A)-infected cells compared to the amounts in lysates of cells infected with vJB31(W596A) or HSV-1(F). This observation was potentially significant, because it was noted previously that mutations precluding scaffold-portal interactions inhibit solubilization of pUL
FIG. 4. pUL6 and scaffold protein interaction in virus-infected cells. CV1 cells were mock infected or infected with HSV-1(F), vJB30(W27A), vJB31(W596A), or a UL6 null mutant. Eighteen hours after infection, coimmunoprecipitation was performed using anti-scaffold (more ...)
To determine whether the mutations affected portal-scaffold interaction as suspected, the clarified lysates were subjected to immunoprecipitation with scaffold-specific antibody. As shown in Fig. , the scaffold-specific antibody readily immunoprecipitated scaffold proteins from all the infected cell lysates. Most importantly, pUL6 coimmunoprecipitated with VP22a from lysates of cells infected with vJB31(W596A), but not from vJB30(W27A)-infected cell lysates (Fig. ). Thus, the inability of vJB30 to replicate in CV1 cells correlated with a failure of its portal and scaffold proteins to interact with infected cells as assessed by immunoprecipitation.
To determine whether the failure of vJB30 scaffold and portal protein interaction affected incorporation of the portal protein into capsids, cells were infected with the UL6 null virus, wild-type HSV-1(F), vJB30(W27A), or vJB31(W596A), and capsids were purified. Capsid proteins were denatured and electrophoretically separated, and an immunoblot of the separated proteins was probed with antibodies to pUL6, scaffold, or VP5 as a loading control. As shown in Fig. , approximately equal amounts of capsids were loaded in each lane, as revealed by the roughly equal intensities of the VP5-specific bands. Similar levels of scaffold proteins were also contained within each lane, suggesting that the portal mutations did not affect scaffold incorporation into capsids. Most significantly, less pUL6-specifc signal was present in the vJB30(W27A) capsids than was detected in wild-type capsids or vJB31(W596A) capsids. To ensure that the association of mutant portal with capsids was not a consequence of the protein migrating throughout the sucrose gradient, all gradient fractions were probed with the pUL6 antibody and VP5 antibody was used to mark capsid-containing fractions. pUL6 immunoreactivity was detected only in fractions containing VP5, indicating that the mutant portal protein did not migrate throughout the sucrose gradient (data not shown). Thus, the failure of vJB30(W27A) portal and scaffold to interact, as revealed by immunoprecipitation, correlated with a reduced, but not complete, inhibition of portal incorporation into vJB30 capsids.
FIG. 5. B-capsid proteins probed with pUL6-, VP22a-, or VP5-specific antibodies. CV1 cells (4 × 108) were infected with 5 PFU/cell of HSV-1(F), UL6 null, vJB30(W27A), or vJB31(W596A). Twenty hours after infection, B capsids were purified, denatured, separated (more ...)
Given the absence of a scaffold/portal interaction in cells infected with vJB30, we predicted that, as in the case of UL6 null viruses, genomic DNA would not be cleaved from concatameric DNA. To test this possibility, viral DNA was purified from cells infected with wild-type HSV-1(F), UL6 null virus, vJB30, vJB30R, or vJB31. The purified DNA was then digested with BamHI, transferred to a nylon membrane, and probed with radiolabeled DNA from HSV-1 genomic ends. The results are shown in Fig. . In cells infected with wild-type virus, the viral cleavage and packaging machinery cleaves sequences corresponding to the BamHI S-P fragment, yielding, upon digestion with BamHI, the BamHI P fragment. This was observed in lanes containing DNA from HSV-1(F)-, vJB30R-, and, to a lesser extent, vJB31(W596A)-infected cells. In contrast to these results, the BamHI P fragment was not observed in BamHI-digested DNA from cells infected with the UL6 null virus or vJB30(W27A). We concluded that the pUL26 W27A mutation precludes DNA cleavage, whereas the W596A mutation does not. Thus, the failure to cleave DNA correlates with the inability of vJB30(W27A) to incorporate portal efficiently into capsids and to propagate in CV1 cells.
FIG. 6. Southern blotting of viral genomes digested with BamHI. CV1 cells (2 × 106) were infected with 5 PFU/cell of HSV-1(F), UL6 null, JB30(W27A), vJB31(W596A), or vJB30R. Fifteen hours after infection, total nuclear DNA was extracted, digested with (more ...)