DNA Topology and the Initiation of Virus DNA Packaging

Abstract

During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase's small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead's portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented.

Fig 1. Elements of cos.

A. Structure of cos^λ. cosN is the site at which TerL endonuclease centers introduce staggered nicks to generate the cohesive ends of λ virion DNA. cosB is the complex site at which TerS binds to anchor TerL: R3, R2 and R1 are TerS binding sites, and I1 is a binding site for the E. coli site-specific DNA bending protein, IHF. I2 is located between cosN and cosB. cosQ is essential for DNA packaging termination. B. Alignment of the I2-containing left DNA ends, i.e., bp 1–70, of λ-like phages N15 (green), λ (black), 21 (blue) and gifsy-1 (red). The I2 segment extends approximately from bp 18 to 50. Approximate positions of R3 segments are underlined. C. The sequence of the left DNA end, bp 1–70, of I2⁺ (above) and I2^re18-50 (below). The I2^re18-50 mutation replaces the AT-rich I2⁺ sequence without changing the cosN-cosB spacing. Underlining highlights the poly-dA and poly-dT segments of I2⁺.

Fig 2. Effect of I2^re mutations on virus yield.

Lysogens of λ-P1 I2⁺ and the I2^re mutants were induced and the phage yields in the resulting lysates were determined. The I2⁺ sequence is shown at the top of the figure in black. The sequences of the replacement mutations are in red. For the lethal I2^re30-35 mutant, the kanamycin-transducing titer was determined. The large I2^re18-29 mutation was used because an earlier study with 6 bp-long mutations indicated that the entire segment could be replaced without affecting virus growth. These data are from a single experiment; a repeat experiment gave equivalent results.

Fig 3. A static bend at I2: permutation analysis of a 150 bp DNA fragment containing λ I1⁺, I2⁺, I2^re30-35, or I2^re18-50.

A. Diagram of the pBend plasmid showing the XbaI and SalI sites used to insert the I2 segments. Flanking the I2 inserts are repeated segments with the restriction enzyme target sites used to generate DNAs with permutations of I2 position. B. Relative mobilities of the permuted DNA fragments versus positions of the I2 segment. Relative mobility 1.0 indicates the mobility of a 150 bp DNA marker. The calculated bend angles [40] are given adjacent to the relevant mobility curve.

Fig 4. Effects of I2 mutations on the production of phage-related structures.

A. Electron micrograph of phage-related structures in a λ-P1 wild type lysate. Symbols: black arrow = prohead; white arrow = intact phage; white chevron = unknown structure. B. Quantitation of phage-related structures in phage lysates. Total numbers of particles observed were: λ-P1 I2⁺—419; λ-P1 I2^re30-35–700; and λ-P1 I2^re18-50–100. Error bars represent standard deviations of data averaged from several EM preparations. For the I2^re18-50 lysate, a single EM preparation was examined.

Fig 5. Effects of I2 mutations on in vivo cos cleavage.

A. Rationale of the in vivo cos cleavage assay. Total phage nucleic acids were isolated from λ-infected cells. AccI digestion of intracellular DNA not cut at cos results in a 7681 bp-long joint DNA fragment (J). cosN cleavage generates 5591 bp right (R) and 2190 bp left (L) end pieces. In an infection by a mutant that is able to cut cos and form complex I, but is unable package DNA, the uncut J and cut L fragments can be detected. AccI-cut total DNA was electrophoresed on a 0.8% agarose gel and transferred to a membrane for southern blotting. B. AccI digested intracellular DNAs: lane 1 is the DNA probe used for Southern blot assay (λ bp 177–2099). Lanes 2 and 3 are AccI-cut λ DNA loaded on the gel before (lane 2) and after heat treatment at 70°C for 10 min to melt the cohesive ends (lane 3). Phage DNAs from positive and negative control phages λ-P1 I2⁺ and λ-P1 cos2 (ΔcosN), respectively are in lanes 4 and 5. In lanes 6, 7, and 8 are DNAs from λ-P1 I2⁺ (a second sample), λ-P1 I2^re30-35 and λ-P1 I2^re18-50, respectively.

Fig 6. Effect of I2 mutations on in vitro cos cleavage.

I2-containing 2.9 kb pOER1-5 DNAs (Table 2), linearized with AatII, were used as cos-cleavage substrates. After heating at 70°C for 10 min, to melt cohesive ends the 0.6 (L) and 2.3 (R) kb reaction products were run on agarose gels and stained with ethidium bromide. Band intensity was measured with a Typhoon phosphoimager. Reactions were done in the presence (left panel) and absence (right panel) of IHF (see Materials and Methods).

Fig 7. Early DNA packaging steps at which I2 might act.

Four steps at which an I2 defect might interrupt DNA packaging are numbered. The step blocked for Aam42 terminase is also indicated. Model 1a: Failure to separate the newly created cohesive ends, followed by dissociation of terminase, and re-ligation that reseals the nicks. Model 1b: Reannealing of the cohesive ends followed by religation. Model 2: I2 mutations block formation of, or destabilize, Complex I. Note that accompanying Complex I formation, the R_end DNA end is released and subject to exonuclease digestion; this is indicated by R_end. Model 3: I2 mutations interfere with proper threading of the DNA through the motor assembly so that the DNA is translocated into the cytoplasm and is subject to exonuclease digestion. DNA represents the nuclease-susceptible virion DNA. The Aam42 defect is the absence of an intact prohead binding domain at the C-terminus of TerL, which prevents Complex I from docking on the portal and assembling an active motor [35, 49, 50].