Analysis Of The Ul97 Phosphotransferase Coding Sequence In Clinical Cytomegalovirus Isolates And Identification Of Mutations Conferring Ganciclovir Resistance

Abstract

The UL97 phosphotransferase coding sequences of clinical cytomegalovirus (CMV) isolates, 10 resistant and 11 sensitive to ganciclovir, were compared to define mutations associated with drug resistance. In each ganciclovir-resistant isolate, a mutation was found that resulted in an amino acid substitution at codon 460 (4 isolates), codon 594 (2 isolates), or codon 595 (4 isolates). No sensitive isolate carried any of these mutations. Marker transfer studies showed that each mutation was capable of conferring ganciclovir resistance to the laboratory CMV strain AD169. Rapid diagnostic tests based on DNA amplification and restriction enzyme analysis were developed for these mutations. Specific mutant DNAs were detected when they constituted at least 10% of the population in the specimen. Several mutations in UL97 appear to be common markers for ganciclovir resistance, and their detection may be a rapid alternative to conventional cell culture susceptibility testing.

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself.

“…Even the lack of 240 aa in rVV228 did not prevent phosphorylation of GCV, which was strongly reduced but still higher than in the mock-infected cell control. The point mutations and the 4 aa deletion (aa 590 to 593) in the Cterminal region, found in GCV-resistant HCMV strains, also led to decreased but not completely abrogated GCV phosphorylation in the vaccinia virus system, as described for the corresponding HCMV variants (Hanson et al, 1995 ;Lurain et al, 1994 ;Chou et al, 1995 ;Sullivan et al, 1992).…”

“…For cloning of point mutations 520H Q (strain CMV-6, a kind gift from A. Erice, University of Minnesota Medical School, MS, USA) (Hanson et al, 1995) and 594A V (Chou et al, 1995) (done in the laboratory of G. Gerna, Servizio di Virologia, Pavia, Italy) in the UL97 ORF of GCV-resistant clinical isolates, the previously described primers pri.5-KpnI and pri.6-EcoRI (Metzger et al, 1994) were used for amplification of an appropriate fragment of DNA from the clinical isolates. The mutation at amino acid position 460M (Lurain et al, 1994) was introduced by site-directed mutagenesis according to the method of Landt et al (1990) by using the mutated primer 5h ATT ACA CCC GTG AAC GTG C 3h for amplification.…”

“…However, we could show that, by using rVV, the quantification of GCV phosphorylation was possible (Michel et al, 1996 ;Zimmermann et al, 1997) in the absence of any other HCMV genes. Hence, we generated a set of rVVs carrying different wild-type or truncated UL97 ORFs, as well as UL97 constructs carrying point mutations which had been identified in GCV-resistant clinical HCMV isolates (Hanson et al, 1995 ;Lurain et al, 1994 ;Chou et al, 1995 ;Sullivan et al, 1992). A few of these UL97 mutants had been used in the previously described transient expression experiments (Michel et al, 1996).…”

“…which induce the resistance of HCMV strains to GCV in biological assays (Sullivan et al, 1992 ;Lurain et al, 1994 ;Wolf et al, 1995 ;Baldanti et al, 1995 ;Chou et al, 1995 ;Hanson et al, 1995). Until now, only point mutations between amino acids 460 and 607 have been detected in GCV-resistant clinical isolates, and neither isolation of clinical HCMV strains lacking the UL97 ORF or carrying extended deletions in the UL97 ORF has been reported, nor has anyone succeeded in generating such mutants in the laboratory (He et al, 1997 ;Michel et al, 1996).…”

Resistance to combined ganciclovir and foscarnet therapy in a liver transplant recipient with possible dual-strain cytomegalovirus coinfection

“…Ganciclovir-resistant mutations in the UL97 phosphotransferase gene are the most widely documented. 9,10 Multidrug resistance has also been correlated with mutations in the UL54 DNA polymerase gene alone or in association with UL97 mutations. Depending on the functional domain in which the mutation occurs, UL54 mutations may result in decreased sensitivity to ganciclovir, cidofovir, or foscarnet.…”