Physical and Functional Characterization of a Viral Genome Maturation Complex

Abstract

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Figure 1

The λ-terminase protomer and IHF cooperatively bind to cosDNA. Protomer binding to radiolabeled DNA (30 pM) was examined using an electrophoretic gel shift assay as described in Materials and Methods. (A) Representative gel shows binding of the protomer to nsDNA in the absence of IHF. (B) Gel shows binding of the protomer to nsDNA in the presence of 5 nM of IHF. Note that IHF does not bind to nsDNA at this concentration (28, 43). (C) Gel shows binding of the protomer to cosDNA in the absence of IHF. (D) Gel shows binding of the protomer to cosDNA in the presence of 5 nM IHF. The concentration of protomer included in each reaction mixture (nM) is indicated at the top of each gel.

Figure 2

The binding data in Fig. 1 (♦, nsDNA minus IHF; ○, cosDNA minus IHF; ●, cosDNA plus IHF) was quantified as described in Materials and Methods. (Solid lines) Shown here is best fit of the data to a phenomenological Hill model, which affords the data presented in Table 1.

Figure 3

SE-AUC analysis of protomer⋅DNA complexes. (A) The binary IHF⋅cosDNA complex was assembled under stoichiometric binding conditions (200 nM cosDNA, 250 nM IHF) and the sample was spun at 6 K (brown), 7.3 K (orange), 9 K (yellow), 12 K (green), and 15 K (blue) rpm. (Solid lines) Global best fit of the data to a single-species model (Eq. 1) is shown, with residuals shown below. The graphic depicts one IHF heterodimer (purple oval) bound to the I1 element in cos to induce a strong bend in the duplex. (B) The ternary protomer⋅IHF⋅cosDNA maturation complex was assembled under stoichiometric binding conditions (200 nM cosDNA, 250 nM IHF, 1 μM protomer) and the sample was spun at 4 K (red), 6 K (orange), 7.3 K (yellow), 9 K (green), and 12 K (blue) rpm. (Solid lines) Given here is global best fit of the data to a single-species model, with residuals shown below. The graphic depicts IHF (purple) and four protomers (blue/cyan) bound at the cos site. (C) The post-cleavage maturation complex was prepared as in (B) except that magnesium was included in the reaction mixture. The sample was spun at 4 K (red), 6 K (orange) and 9 K (yellow) rpm. (Solid lines) Given here is the global best fit of the data to a noninteracting, two-species model (Eq. 3), with residuals shown below. The graphic depicts IHF (purple) and four protomers (blue/cyan) bound at the matured genome end (D_L) and the upstream D_R duplex that has been ejected from the complex. To see this figure in color, go online.

Figure 4

Stoichiometry of protomer and IHF in the nucleoprotein complexes. The λ-protomer was added to cosDNA in the absence (○) or the presence (●) of IHF as described in Materials and Methods, except that 400 nM DNA was used in the former case. The σ-value at each protomer/DNA was determined by SE-AUC according to a single-species model (Eq. 1), which was used to determine M_B according to Eq. 2. Each data point represents the derived M_B with error bars indicating the 68% confidence intervals. (Solid lines) Given here is the best fit of each data set to a stoichiometric binding model (Eq. 5).

Figure 5

Cos-cleavage nuclease activity. (A) Stoichiometric complexes were assembled under conditions identical to those used in the AUC studies (200 nM cosDNA, 1 μM protomer) in the absence (○) and presence (●) of 250 nM IHF. Mg²⁺ was then added to initiate the cos-cleavage reaction. Each data point represents the average of three independent experiments with error bars indicating the SDs. (B) The cos-cleavage reaction was conducted as described in Materials and Methods using 30 pM radiolabeled DNA, 5 nM IHF, and the indicated concentration of protomer. The reaction products were analyzed by agarose gel, which reveals the presence of two products—the retarded maturation complex composed of terminase bound to the matured D_L strand and the upstream D_R strand that has been ejected from the complex. Lane F depicts free DNA in the absence of protein. IHF was included in all other samples and the concentration of protomer included in each reaction is indicated at the top of the gel. Lane Δ depicts a reaction mixture containing 150 nM protomer that was heated to 70°C for 5 min before loading on the gel. (Right) Migration of the intact cos274 substrate, the binary IHF⋅DNA complex, and the D_L and D_R nuclease products are indicated here.

Figure 6

Assembly of a functional maturation complex in the infected cell. (Red) Characterized binding and self-assembly interactions are shown with known dissociation constants. Based on these thermodynamic constants and the presumed concentrations of IHF and protomer in the infected cell, the preferred packaging pathway in vivo is indicated (black arrows). We propose that the tetrameric protomer stoichiometry observed in the post-cleavage complex is retained in the λ-packaging motor, as depicted. Details are discussed in the text. To see this figure in color, go online.