Rapid genetic engineering of human cytomegalovirus by using a lambda phage linear recombination system: demonstration that pp28 (UL99) is essential for production of infectious virus

Abstract

A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

FIG. 1.

Construction of UL99 recombinant BACs. The genome of HCMV AD169 is shown at the top with the UL99 ORF and its coordinates listed below. This AD169 BAC was transferred to E. coli strain DY380 for these experiments (25). The position of insertion of the Amp^r lacZ cassette in the UL99 deletion BAC (99KO) is shown. The UL99 deletion BAC, 99KO, was then repaired with 800-bp linear PCR products encoding either wild-type sequences (99Rev) or mutant sequences (99STOP and 99ΔMyr) by previously described methods (10, 25, 26). The sequences containing the point mutations in BACs 99STOP and 99ΔMyr along with the corresponding sequence of the wild-type repaired BAC, 99Rev, are shown at the bottom, with changes in boldface italics.

FIG. 2.

Analysis of 99KO, 99STOP, and 99ΔMyr BAC by Southern hybridization. (A) DNA prepared from three independent blue, ampicillin-resistant colonies of BAC-containing E. coli that had been recombined with the linear Amp^r lacZ cassette were digested with HindIII, subjected to electrophoresis in agarose gel, and transferred to nitrocellulose membranes. Hybridization was carried out with a ³²P-labeled probe generated by nick translation of DNA containing the Amp^r lacZ cassette. Arrow, migration of the approximately 8-kbp HindIII fragment containing the Amp^r lacZ cassette. Note that a single fragment hybridized with the probe, indicating a single site of insertion. (B) DNA prepared from E. coli containing the wild-type AD169 BAC (AD169), 99KO BAC, mutant 99STOP BAC, and mutant 99ΔMyr BACs was digested with HindIII and analyzed as described for panel A by Southern hybridization with a ³²P-labeled probe generated by nick translation of DNA containing the intact UL99 ORF. Arrow, migration of the HindIII R fragment at approximately 6 kbp. The weak signal in the lane containing the 99KO DNA is secondary to the approximate 100 bp of the 3′ end of the UL99 ORF that remains following this insertion and deletion.

FIG. 3.

PCR analysis of 99KO, 99STOP, 99ΔMyr, and the wild-type repaired (99Rev) BACs. (A) DNA from 99KO, 99STOP, or 99ΔMyr BACs was amplified by primer pairs used to generate the recombination cassette, and the resulting products were analyzed by agarose gel electrophoresis. The expected product from the wild-type parent BAC (AD169) was approximately 800 bp. The amplimers from the 99STOP and 99ΔMyr mutant BACs migrated similarly, whereas the amplimer of the 99KO BAC was approximately 1.2 kbp larger. (B) DNA from the wild-type parent BAC (AD169), the 99KO BAC, and the wild-type repaired BAC, 99Rev, was amplified as described for panel A, and the amplimers were analyzed by agarose gel electrophoresis. The amplimers of the wild-type parent BAC and the wild-type repaired BAC migrated at approximately 800 bp.