Existence of transdominant and potentiating mutants of UL9, the herpes simplex virus type 1 origin-binding protein,suggests that levels of UL9 protein may be regulated during infection

UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection.     

Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein.

UL9, the origin-binding protein of herpes simplex virus type 1 (HSV-1), has been overexpressed in an insect cell overexpression system and purified to homogeneity. In this report, we confirm and extend recent findings on the physical properties, enzymatic activities, and binding properties of UL9. We demonstrate that UL9 exists primarily as a homodimer in solution and that these dimers associate to form a complex nucleoprotein structure when bound to the HSV origin of replication. We also show that UL9 is an ATP-dependent helicase, capable of unwinding partially duplex DNA in a sequence-independent manner. Although the helicase activity of UL9 is demonstrable on short duplex substrates in the absence of single-stranded DNA-binding proteins, the HSV single-stranded DNA-binding protein ICP8 (but not heterologous binding proteins) stimulates UL9 to unwind long DNA sequences of over 500 bases. We were not able to demonstrate unwinding of fully duplex DNA sequences containing the HSV origin of replication. However, in experiments designed to detect origin-dependent unwinding, we did find that UL9 wraps supercoiled DNA independent of sequence or ATP hydrolysis.     

Activation of the herpes simplex virus type-1 origin-binding protein (UL9) by heat shock proteins.

Heat shock proteins participate in the initiation of DNA replication of different organisms by facilitating the assembly of initiation complexes. We have examined the effects of human heat shock proteins (Hsp40 and Hsp70) on the interaction of the herpes simplex virus type-1 initiator protein (UL9) with oriS, one of the viral origins of replication. Hsp40 and Hsp70 act substoichiometrically to increase the affinity of UL9 for oriS. The major contributor to this effect is Hsp40. Heat shock proteins also stimulate the ATPase activity of UL9 with oriS and increase opening of the origin. In contrast, heat shock proteins have no effect on the origin-independent activities of UL9 suggesting that their role is not merely in refolding denatured protein. These observations are consistent with a role for heat shock proteins in activating UL9 to efficiently initiate viral origin-dependent DNA replication. The action of heat shock proteins in this capacity is analogous to their role in activating the initiator proteins of other organisms.     

The conserved helicase motifs of the herpes simplex virus type 1 origin-binding protein UL9 are important for function.

The UL9 gene of herpes simplex virus encodes a protein that specifically recognizes sequences within the viral origins of replication and exhibits helicase and DNA-dependent ATPase activities. The specific DNA binding domain of the UL9 protein was localized to the carboxy-terminal one-third of the molecule (H. M. Weir, J. M. Calder, and N. D. Stow, Nucleic Acids Res. 17:1409-1425, 1989). The N-terminal two-thirds of the UL9 gene contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases, suggesting that this region may be important for helicase activity of UL9. In this report, we examined the functional significance of these six motifs for the UL9 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. An in vivo complementation test was used to study the effect of each mutation on the function of the UL9 protein in viral DNA replication. In this assay, a mutant UL9 protein expressed from a transfected plasmid is used to complement a replication-deficient null mutant in the UL9 gene for the amplification of herpes simplex virus origin-containing plasmids. Mutations in five of the six conserved motifs inactivated the function of the UL9 protein in viral DNA replication, providing direct evidence for the importance of these conserved motifs. Insertion mutants resulting in the introduction of two alanines at 100-residue intervals in regions outside the conserved motifs were also constructed. Three of the insertion mutations were tolerated, whereas the other five abolished UL9 function. These data indicate that other regions of the protein, in addition to the helicase motifs, are important for function in vivo. Several mutations result in instability of the mutant products, presumably because of conformational changes in the protein. Taken together, these results suggest that UL9 is very sensitive to mutations with respect to both structure and function, perhaps reflecting its multifunctional character.     

Herpes simplex virus origin-binding protein (UL9) loops and distorts the viral replication origin.

To investigate the role of the herpes simplex virus origin-binding protein (UL9) in the initiation of DNA replication, we have examined the effect of UL9 binding on the structure of the viral origin of replication. UL9 loops and alters the DNA helix of the origin regardless of the phasing of the binding sites. DNase I and micrococcal nuclease footprinting show that UL9 binds two sites in the origin and loops the AT-rich DNA between them independent of the topology of the DNA. KMnO4 and dimethyl sulfate footprinting further show that UL9 alters the DNA helix in the AT region. In contrast to the looping reaction, however, helical distortion requires the free energy of supercoiled DNA. UL9 also loops and distorts the origin DNA of a replication-defective mutant with a 6-bp insertion in the AT region. Because the helical distortion of this mutant DNA is different from that of functional origins, we conclude that an imperfect tertiary structure of the mutant DNA may contribute to its loss of replication function.     

Helicase motif Ia is involved in single-strand DNA-binding and helicase activities of the herpes simplex virus type 1 origin-binding protein,UL9

UL9 is a multifunctional protein essential for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. It is thought that UL9 binds the origin of replication and unwinds it in the presence of ATP and the HSV-1 single-stranded DNA (ssDNA)-binding protein. We have previously characterized the biochemical properties of mutants in all helicase motifs except for motif Ia (B. Marintcheva and S. Weller, J. Biol. Chem. 276:6605-6615, 2001). Structural information for other superfamily I and II helicases indicates that motif Ia is involved in ssDNA binding. By analogy, we hypothesized that UL9 motif Ia is important for the ssDNA-binding function of the protein. On the basis of sequence conservation between several UL9 homologs within the Herpesviridae family and distant homology with helicases whose structures have been solved, we designed specific mutations in motif Ia and analyzed them genetically and biochemically. Mutant proteins with residues predicted to be involved in ssDNA binding (R112A and R113A/F115A) exhibited wild-type levels of intrinsic ATPase activity and moderate to severe defects in ssDNA-stimulated ATPase activity and ssDNA binding. The S110T mutation targets a residue not predicted to contact ssDNA directly. The mutant protein with this mutation exhibited wild-type levels of intrinsic ATPase activity and near wild-type levels of ssDNA-stimulated ATPase activity and ssDNA binding. All mutant proteins lack helicase activity but were able to dimerize and bind the HSV-1 origin of replication as well as wild-type UL9. Our results indicate that residues from motif Ia contribute to the ssDNA-binding and helicase activities of UL9 and are essential for viral growth. This work represents the successful application of an approach based on a combination of bioinformatics and structural information from related proteins to deduce valuable information about a protein of interest.     

The interaction of herpes simplex type 1 virus origin-binding protein (UL9 protein) with Box I, the high affinity element of the viral origin of DNA replication.

The herpes simplex type 1 (HSV-1) origin binding protein, the UL9 protein, exists in solution as a homodimer of 94-kDa monomers. It binds to Box I, the high affinity element of the HSV-1 origin, Oris, as a dimer. The UL9 protein also binds the HSV-1 single strand DNA-binding protein, ICP8. Photocross-linking studies have shown that although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I. It is this form of the UL9 homodimer that upon interaction with ICP8, promotes the unwinding of Box I coupled to the hydrolysis of ATP to ADP and Pi. Photocross-linking studies have also shown that the amount of UL9 protein that interacts with Box I is reduced by its interaction with ICP8. Antibody directed against the C-terminal ten amino acids of the UL9 protein inhibits its Box I unwinding activity, consistent with the requirement for interaction of the C terminus of the UL9 protein with ICP8. Inhibition by the antibody is enhanced when the UL9 protein is first bound to Box I, suggesting that the C terminus of the UL9 protein undergoes a conformational change upon binding Box I.     

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain.

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.     

Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.     

Functional interaction between the herpes simplex virus type 1 polymerase processivity factor and origin-binding proteins: enhancement of UL9 helicase activity

The origin (ori)-binding protein of herpes simplex virus type 1 (HSV-1), encoded by the UL9 open reading frame, has been shown to physically interact with a number of cellular and viral proteins, including three HSV-1 proteins (ICP8, UL42, and UL8) essential for ori-dependent DNA replication. In this report, it is demonstrated for the first time that the DNA polymerase processivity factor, UL42 protein, provides accessory function to the UL9 protein by enhancing the 3'-to-5' helicase activity of UL9 on partially duplex nonspecific DNA substrates. UL42 fails to enhance the unwinding activity of a noncognate helicase, suggesting that enhancement of unwinding requires the physical interaction between UL42 and UL9. UL42 increases the steady-state rate for unwinding a 23/38-mer by UL9, but only at limiting UL9 concentrations, consistent with a role in increasing the affinity of UL9 for DNA. Optimum enhancement of unwinding was observed at UL42/UL9 molecular ratios of 4:1, although enhancement was reduced when high UL42/DNA ratios were present. Under the assay conditions employed, UL42 did not alter the rate constant for dissociation of UL9 from the DNA substrate. UL42 also did not significantly reduce the lag period which was observed following the addition of UL9 to DNA, regardless of whether UL42 was added to DNA prior to or at the same time as UL9. Moreover, addition of UL42 to ongoing unwinding reactions increased the steady-state rate for unwinding, but only after a 10- to 15-min lag period. Thus, the increased affinity of UL9 for DNA most likely is the result of an increase in the rate constant for binding of UL9 to DNA, and it explains why helicase enhancement is observed only at subsaturating concentrations of UL9 with respect to DNA. In contrast, ICP8 enhances unwinding at both saturating and subsaturating UL9 concentrations and reduces or eliminates the lag period. The different means by which ICP8 and UL42 enhance the ability of UL9 to unwind DNA suggest that these two members of the presumed functional replisome may act synergistically on UL9 to effect initiation of HSV-1 DNA replication in vivo.     

Genome replication and progeny virion production of herpes simplex virus type 1 mutants with temperature-sensitive lesions in the origin-binding protein

Genome replication of herpes simplex viruses (HSV) in cultured cells is thought to be started by the action of the virus-encoded origin-binding protein (OBP). In experiments using two HSV-1 mutants with temperature-sensitive lesions in the helicase domain of OBP, we demonstrated that this function is essential during the first 6 hours of the lytic cycle. Once DNA synthesis has started, this function is no longer required, suggesting that origin-driven initiation of viral DNA replication is a single event rather than a continuous process.     

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.     

Varicella-zoster virus gene 51 complements a herpes simplex virus type 1 UL9 null mutant.

Varicella-zoster virus (VZV) gene 51 encodes a protein which is homologous to UL9, the origin of DNA replication-binding protein of herpes simplex virus type 1. No genetic information is available on VZV gene 51, but its product has been shown to bind to virtually the same recognition sequence as does UL9 (D. Chen and P. D. Olivo, J. Virol. 68:3841-3849, 1994; N. D. Stow, H. M. Weir, and E. C. Stow, Virology 177:570-577, 1990). We report here that gene 51 can complement a UL9 null mutant (hr94) (A. K. Malik, R. Martinez, L. Muncy, E. P. Carmichael, and S. K. Weller, Virology 190:702-715, 1992), but at a level which is only 20% of that of UL9. Quantitation of viral DNA synthesis suggests that this phenotype is due to a defect in viral DNA synthesis. Regardless, the ability of VZV gene 51 to complement UL9 suggests that alphaherpesviruses have a highly conserved mechanism of initiation of viral DNA synthesis.     

Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains.

Syncytial (syn) mutants of herpes simplex virus cause cell fusion. Many syn mutations map to the syn1 locus, which has been identified with the gK (UL53) gene. In this work, the gK genes of eight syn mutants derived from the KOS strain were sequenced to identify residues and, possibly, domains important for the fusion activity of mutant gK. DNA sequencing showed that six mutants (syn30, syn31, syn32, syn102, syn103, and syn105) had single missense mutations in the gK gene. Two of these, syn31 and syn32, had identical mutations that caused the introduction of a potential site for N-linked glycosylation. syn31 gK was analyzed by in vitro translation and found to utilize the novel glycosylation site. Two other mutants, syn8 and syn33, had three mutations each, resulting in three amino acid substitutions in syn8 and two substitutions in syn33. Of the 10 gK syn mutant sequences known, 8 have mutations in the N-terminal domain of gK, suggesting that this domain, which is likely to be an ectodomain, is important for the function of the protein. The other two mutants, syn30 and syn103, have mutations near the C terminus of gK.     

Packaging determinants in the UL11 tegument protein of herpes simplex virus type 1

The UL11 gene of herpes simplex virus type 1 encodes a 96-amino-acid tegument protein that is myristylated, palmitylated, and phosphorylated and is found on the cytoplasmic faces of nuclear, Golgi apparatus-derived, and plasma membranes of infected cells. Although this protein is thought to play a role in virus budding, its specific function is unknown. Purified virions were found to contain approximately 700 copies of the UL11 protein per particle, making it an abundant component of the tegument. Moreover, comparisons of cell-associated and virion-associated UL11 showed that packaging is selective for underphosphorylated forms, as has been reported for several other tegument proteins. Although the mechanism by which UL11 is packaged is unknown, previous studies have identified several sequence motifs in the protein that are important for membrane binding, intracellular trafficking, and interaction with UL16, another tegument protein. To ascertain whether any of these motifs are needed for packaging, a transfection/infection-based assay was used in which mutant forms of the protein must compete with the wild type. In this assay, the entire C-terminal half of UL11 was found to be dispensable. In the N-terminal half, the sites of myristylation and palmitylation, which enable membrane-binding and Golgi apparatus-specific targeting, were found to be essential for efficient packaging. The acidic cluster motif, which is not needed for Golgi apparatus-specific targeting but is involved in recycling the protein from the plasma membrane and for the interaction with UL16, was found to be essential, too. Thus, something other than mere localization of UL11 to Golgi apparatus-derived membranes is needed for packaging. The critical factor is unlikely to be the interaction with UL16 because other mutants that fail to bind this protein (due to removal of the dileucine-like motif or substitutions with foreign acidic clusters) were efficiently packaged. Collectively, these results suggest that UL11 packaging is not driven by a passive mechanism but instead requires trafficking through a specific pathway.     

Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.     

Binding partners for the UL11 tegument protein of herpes simplex virus type 1

The product of the U(L)11 gene of herpes simplex virus type 1 (HSV-1) is a 96-amino-acid tegument protein that accumulates on the cytoplasmic face of internal membranes. Although it is thought to be important for nucleocapsid envelopment and egress, the actual function of this protein is unknown. Previous studies focused on the characterization of sequence elements within the UL11 protein that function in membrane binding and trafficking to the Golgi apparatus. Binding was found to be mediated by two fatty acyl groups (myristate and palmitate), while an acidic cluster and a dileucine motif were identified as being important for the recycling of UL11 from the plasma membrane to the Golgi apparatus. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Using both immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we identified a 40-kDa protein that specifically associates with UL11 from infected Vero cells. Mutational analyses revealed that the acidic cluster and the dileucine motif are required for this association, whereas the entire second half of UL11 is not. In addition, UL11 homologs from pseudorabies and Marek's disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. Purification and analysis of the 40-kDa protein by mass spectrometry revealed that it is the product of the U(L)16 gene, a virion protein reported to be involved in nucleocapsid assembly. Cells transfected with a UL16-green fluorescent protein expression vector produced a protein that was of the expected size, could be pulled down with GST-UL11, and accumulated in a Golgi-like compartment only when coexpressed with UL11, indicating that the interaction does not require any other viral products. These data represent the first steps toward elucidating the network of tegument proteins that UL11 links to membranes.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA

The herpes simplex virus type 1 UL42 DNA polymerase processivity factor interacts physically with UL9 and enhances its ability to unwind short, partially duplex DNA. In this report, ATP hydrolysis during translocation of UL9 on single-stranded (ss) or partially duplex DNA was examined in the presence and absence of UL42 to determine the effect of UL42 on the catalytic function of UL9. Our studies reveal that a homodimer of UL9 is sufficient for DNA translocation coupled to ATP hydrolysis, and the steady-state ATPase catalytic rate was greater on partially duplex DNA than on ss DNA in the presence or absence of UL42. Although UL42 protein increased the steady-state rate for ATP hydrolysis by UL9 during translocation on either partially duplex or ss DNA, UL42 had no significant effect on the intrinsic ATPase activity of UL9. UL42 also had no effect on the catalytic rate of ATP hydrolysis when UL9 was not limiting but enhanced the steady-state ATPase rate at only subsaturating UL9 concentrations. At subsaturating UL9 to DNA ratios, stoichiometric concentrations of UL42 were shown to increase the amount of UL9 bound to ss DNA at equilibrium. These data support a model whereby UL42 increases the ability of UL9 to load onto DNA, thus increasing its ability to assemble into a functional complex capable of unwinding duplex DNA.     

Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1

By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 A, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.