Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection. Human papillomaviruses (HPVs)4 are non-enveloped small DNA viruses of great medical importance. Among the large group of HPVs known by now, sexually transmitted genital high risk HPV types are the cause for the development of a variety of epithelial tumors, especially cervical carcinoma (1). Cervical cancer is the second leading cause of death among female cancer patients worldwide. HPV16 and HPV18 stand out, as they are causally linked to Ͼ70% of cervical cancer cases (2).HPV particles consist of 72 pentamers of the major capsid protein L1, which forms the virus outer shell and encapsidates the viral DNA (3, 4). The minor capsid protein L2 is present at up to 72 copies and is hidden inside the capsid with exception of a small N-terminal section (5, 6). Efficient infection by HPV16 and HPV18 pseudoviruses requires the interactions of the L1 protein with extracellular matrix (ECM)-and cell surface-resident heparan sulfate receptor in vitro (7-9) as well as in vivo models (10). Homologs of heparan sulfate polysaccharide or heparin, secreted by mast cells, can inhibit HPV infection (7-9).Cell-surface heparan sulfates are linear and highly negatively charged oligosaccharides that are covalently linked to proteins. They can serve as the attachment receptors for several important human virus pathogens (7,11,12). Despite considerable efforts, the interactions between HPV and the heparan sulfate oligosaccharides that initiate infection are poorly understood. Here, we determined the co-crystal structure of HPV16 and HPV18 capsids bound to oligomeric heparin. We found that the highly negatively charged heparin fragment binds to multiple locations on the capsid surface mainly through charge-charge interactions. On the basis of the structure, we generated mutant virus to disrupt the interactions with heparin. ECM and cell binding assays combined with infectivity measurements showed that substitution of key HPV residues involved in bi...

Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.

SummaryTerminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme -DNA maturation and DNA packaging -are discussed.

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1⌬K100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that Ϸ30 amino acids of gpNu1⌬K100 are solvent-exposed and highly flexible. We therefore constructed gpNu1⌬E68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1⌬E68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1⌬E68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1⌬E68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal Ϸ30 residues in gpNu1⌬K100 adopt a pseudo-stable ␣-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.Terminase enzymes are common to many of the doublestranded DNA bacteriophage and eukaryotic DNA viruses such as adenovirus and the herpesvirus groups (2-4). These enzymes function to insert a viral genome into the confines of a preformed, empty capsid. The terminase enzyme from bacteriophage is composed of two virally encoded proteins, gpNu1 (181 amino acids) and gpA (640 amino acids), in a gpA 1 ⅐gpNu1 2 holoenzyme complex (5, 6). Terminase holoenzyme possesses site-specific nuclease (6 -9), ATPase (10 -13), DNA strand separation (9, 14), and DNA translocase (15, 16) catalytic activities that work in concert to package viral DNA. All of the terminase enzymes characterized to date possess a similar holoenzyme composition (small and large subunits) and catalytic activities (5, 17-21).Replication of DNA proceeds through a rolling circle mechanism that gives rise to linear concatemers of the viral genome linked in a head to tail fashion (22, 23). Packaging of viral DNA requires the excision of an individual genome from the concatemer, and packaging of the 48.5-kb 1 duplex within the capsid. Genome packaging by terminase has been described in detail (21, 24 -27) and is summarized here. Packaging initiates with the assembly of the holoenzyme at a cos site in the concatemer. This site represents the junction between the left and right ends of individual genomes within the concatemer (Fig. 1A). Site-specific assem...