Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

Human cytomegalovirus (HCMV), a member of the beta-herpesvirus family, establishes lifelong latent infections in its hosts and is known to manipulate a wide range of cellular processes. A key viral protein involved in host interaction is the kinase pUL97, encoded by the UL97 gene, which is essential for viral replication. Kinase-deficient pUL97 mutants exhibit profound replication defects.
To study pUL97 function more precisely, a modified analog-sensitive variant of the kinase was recently developed. This mutant version can be selectively inhibited by a non-hydrolysable ATP analog, with kinase activity restored upon removal of the inhibitor via media exchange. We incorporated this analog-sensitive pUL97 into the laboratory strain AD169 of HCMV (BADwt), generating the mutant virus BAD-UL97-as1.
In the absence of the ATP analog, BAD-UL97-as1 replicated 1-NM-PP1 comparably to the wild-type virus and retained the ability to phosphorylate cellular targets. However, upon treatment with the ATP analog, the mutant virus exhibited significant defects in both intracellular and extracellular viral DNA production, as well as impaired generation of infectious progeny. Notably, exposure to 3MB-PP1, a selective inhibitor of the analog-sensitive pUL97, led to a marked reduction in phosphorylation of a known pUL97 substrate as early as 4 hours post-treatment. This rapid and reversible inhibition enables functional studies of pUL97 without the confounding effects of reduced viral titers.
However, off-target effects of 3MB-PP1 on various cellular pathways were observed and should be taken into account when using this system.