The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics

Abstract

An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RV⌬UL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E

“…The reason for this is unknown, but it seems likely that it was caused by an illegitimate recombination event during the insertion of the BAC vector by homologous recombination in fibroblasts. Interestingly, a similar unanticipated deletion was found in the HCMV FIXBAC (Murphy et al, 2003b), which was constructed using the same BAC vector and flanking homologous arms (Hahn et al, 2002). With regard to previously reported TB40/E-variants, TB40-BAC4 resembles the Bart strain in that UL141 has a frameshift insertion at codon 63 (Tomasec et al, 2005); however, unlike strain Bart, UL144 and UL145 are intact and thus TB40-BAC4 is identical to the TB40/E sequence published by Dolan et al (2004) in all three genes.…”

“…The EC-propagated HCMV strain TB40/E was cloned as a BAC in Escherichia coli as described previously (Hahn et al, 2002). Briefly, 10 7 HFF were transfected with 35 mg plasmid pEB1097 containing a tk-gpt-bac-cassette flanked with HCMV homologous sequences of US1-US2 (AD169 nt 192 648-193 360; GenBank accession no.…”

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

“…BADwt (47) is derived from a BAC clone of the AD169 HCMV strain; BADrUL131 (19,22) is a derivative of BADwt in which the UL131 ORF has been repaired; BFXwt (48,49) is derived from a BAC clone of the VR1814 clinical HCMV isolate. Viruses were prepared by electroporation of BAC DNAs into HFFs, and the resulting virus preparation was amplified once in ARPE-19 cells or HFFs, unless otherwise specified.…”

Human cytomegalovirus encodes a highly specific RANTES decoy receptor

“…The AD169 laboratory strain (17), ADwt, and the VR1814 clinical isolate (18,19), FIXwt, served as wild-type viruses in these studies. The pUL21.5-deficient mutant virus, ADsubUL21.5, was generated from ADwt by homologous recombination within infected fibroblasts.…”

“…The HCMV sequence from base pair 27105 to 27611 was substituted with a marker cassette containing the GFP under control of an SV40 promoter, followed by the internal ribosomal entry site and a puromycin resistance gene. A second UL21.5-null mutant was prepared from a VR1814-containing bacterial artificial chromosome (BAC), termed FIX BAC (19), which was modified by replacing the guanine phosphoribosyltransferase gene in the BAC sequence with a GFP sequence (S. Terhune, D. Yu, and T.S., unpublished data). The FIX substitution mutant, FIXsubUL21.5, was constructed by linear recombination in Escherichia coli (20).…”