Faculty Bibliography

Drug resistant strains of human cytomegalovirus (HCMV) in patients at risk may increasingly develop into a problem in the clinical setting. Genotypic resistance testing is becoming the method of choice, but requires previous phenotypic characterisation of each newly found mutation. In order to facilitate the interpretation of the patient's CMV sequence data, a web-based search tool was generated that links the sequence to a database containing all published UL97 (protein kinase) and UL54 (DNA polymerase) mutations and corresponding antiviral drug susceptibility phenotypes. It is reasonable to assume that HCMV drug resistance testing will provide relevant data for an adjustment of therapy and on prognosis of clinical outcome. HCMV drug susceptibility testing will become even more important once new drugs will be available for therapy allowing a wider choice of antiviral agents to treat HCMV disease. These topics will also play a pivotal role for optimising antiviral therapy of HCMV- and other viral diseases.

One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator gene, enhanced green fluorescence protein was C-terminally fused to the HCMV early-late protein UL83 (pp65) or the late proteins UL32 (pp150) and UL99 (pp28), resulting in reporter viruses vTB65g, vTB150g, and vTB28g. vTB65g proved to be superior to the other constructs due to its favorable signal-to-noise ratio and was therefore used to establish the optimum conditions for our assay. The UL54 E756K and D413E mutations were introduced into vTB65g by markerless bacterial artificial chromosome mutagenesis, resulting in virus strains vE756Kg and vD413Eg. The drug susceptibility phenotypes of vE756Kg and vD413Eg were comparable to those previously reported. Furthermore, we found a reduced replicative fitness of vE756Kg by measuring fluorescence intensity as well as by conventional virus growth kinetics. Decreased fluorescence signals of vE756Kg- and vD413Eg-infected cells at late times of infection suggested a reduced polymerase activity, which was confirmed by real-time PCR quantification of the newly synthesized viral DNAs. This new fluorescence-based assay is a highly reproducible method for the phenotypic characterization of mutations potentially influencing drug susceptibility, viral replicative fitness, and polymerase activity of HCMV after marker transfer.

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.

Amycolatopsis balhimycina DSM5908 is the producer of the vancomycin-type glycopeptide antibiotic balhimycin. Balhimycin production is controlled by transcriptional regulators and depends on different environmental factors. To analyse the regulatory network P(tba), a representative promoter of the balhimycin gene cluster, was fused to the reporter gene egfp and integrated into the genome of A. balhimycina. Balhimycin production, fluorescence intensity and the amount of tba-transcript were determined under different cultivation conditions in microtiter plates. Thus we could demonstrate that these values are directly correlated. This approach facilitates the analysis of a large number of samples in parallel and it provides the opportunity to easily define conditions to increase balhimycin production.