Analysis of herpes simplex virus type 1 DNA packaging signal mutations in the context of the viral genome

Abstract

The minimal signal required for the cleavage and packaging of replicated concatemeric herpes simplex virus type 1 (HSV-1) DNA corresponds to an approximately 200-bp fragment, Uc-DR1-Ub, spanning the junction of the genomic L and S segments. Uc and Ub occupy positions adjacent to the L and S termini and contain motifs (pac2 and pac1, respectively) that are conserved near the ends of other herpesvirus genomes. We have used homologous Red/ET recombination in Escherichia coli to introduce wild-type and specifically mutated Uc-DR1-Ub fragments into an ectopic site of a cloned HSV-1 genome from which the resident packaging signals had been previously deleted. The resulting constructs were transfected into mammalian cells, and their abilities to replicate and become encapsidated, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus were assessed. In general, the results obtained agree well with previous observations made using amplicons and confirm roles for the pac2 T element in the initiation of DNA packaging and for the GC-rich motifs flanking the pac1 T element in termination. In contrast to a previous report, the sequence of the DR1 element was also crucial for DNA packaging. Following repair of the resident packaging signals in mammalian cells, recombination occurred at high frequency in progeny virus between the repaired sequences and mutated Uc-DR1-Ub inserts. This restored the ability of mutated Uc-DR1-Ub inserts to generate terminal fragments, although these were frequently larger than expected from simple repair of the original lesion.

FIG. 1.

Structure of the HSV-1 Uc-DR1-Ub element. (a) Structure of the HSV-1 genome showing the positions and relative orientations (horizontal arrows) of copies of the a sequence. (b) Circularization of linear genomes by direct ligation brings together two copies of the a sequence separated by a single DR1 repeat. The site of ligation, and of cleavage of concatemers, is shown by the vertical arrow. (c) Motifs and regions within the 194-bp Uc-DR1-Ub fragment. To facilitate naming of mutants, component regions of Uc, Ub, and DR1 were also referred to as c1 to c4, b1 to b4, and R, respectively, as indicated in parentheses.

FIG. 2.

Generation of recombinant HSV-1 BACs carrying the Uc-DR1-Ub fragment. The upper part of the figure depicts the BAC fHSVΔpac, which consists of a complete HSV-1 strain 17 genome, from which all copies of the a sequence have been deleted (indicated as Δa), cloned into the vector pBeloBac11 (22). The lower part shows an expansion of the TK locus indicating the positions of flanking BamHI and NheI sites (coordinates taken from reference 14). Uc-DR1-Ub fragments (black box) and the 1.3-kbp rpsL-neo fragment (white box) were inserted separately into the unique SacI and BspEI sites (positions 47358 and 47174, respectively) within a cloned copy of the BamHI P fragment (positions 45055 to 48634). Recombinant BACs were generated by Red/ET recombination following the introduction of the resulting BamHI fragments into bacteria containing fHSVΔpac. The dotted line shows the position at which replicated concatemeric DNA would be cleaved, and the sizes of the predicted terminal fragments are indicated (kbp). The two possible orientations of the Uc-DR1-Ub fragment are designated + and −, as indicated, and allow identification of genomic fragments terminating in Ub or Uc. For example, the 1.4-kbp BamHI fragment from the + orientation and the 3.8-kbp BamHI fragment from the − orientation each represent Uc-containing termini.

FIG. 3.

Sequences of the HSV-1 Uc-DR1-Ub fragment and of the mutants used in this study. The sequence of the wt fragment (W) is depicted on two lines with the characteristic motifs and regions shown. The site of cleavage of concatemers lies at the right end of the DR1 sequence (6). The substitutions and deletions in the various mutants are shown below the wt sequence. Modified nucleotides are in lowercase, “x” indicates a deleted nucleotide, and “.” indicates an unchanged nucleotide. Only the ΔB mutant is affected at positions within both lines of the wt sequence.

FIG. 4.

Replication and packaging of BACs containing or lacking the Uc-DR1-Ub fragment. BHK cells were transfected with the indicated BAC, and total and DNase-resistant DNAs were prepared. Samples were cleaved with a combination of NheI, NdeI, and DpnI; fractionated by agarose gel electrophoresis; blotted; and hybridized to labeled pGX153 DNA. Lane M contains molecular size markers of the indicated sizes (kbp).

FIG. 5.

Replication and packaging of BACs containing mutated Uc-DR1-Ub fragments. BHK cells were transfected with the indicated BACs, and samples of total (a) and DNase-resistant (b) DNA were analyzed as described for Fig. 4. Lanes M1 and M2 contain molecular size markers whose sizes (kbp) are indicated.

FIG. 6.

Replication and packaging of BACs containing mutated Uc-DR1-Ub fragments. BHK cells were transfected with the indicated BAC, and samples of total and DNase-resistant DNA were analyzed as described for Fig. 4. Lane M contains molecular size markers of the indicated sizes (kbp).

FIG. 7.

Analysis of DNase-resistant DNA from cells infected with rescued progeny. BHK cell monolayers were infected with virus stocks derived from cells cotransfected with the indicated BAC and pGX8 DNA or, in the case of W−, with BAC DNA alone. DNA-resistant DNA was isolated and, following cleavage with BamHI, was analyzed as described for Fig. 4. Lane M contains molecular size markers of the indicated sizes (kbp), and the positions of the 3.8- and 1.4-kbp terminal fragments and 5.2-kbp uncleaved fragment are indicated on the right side.