Heaton, Phillip

ABSTRACT Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54 / UL97 associated with antiviral resistance. Plasma samples ( n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

Human papillomaviruses (HPVs) replicate in the nucleus and modulate several host cell pathways to create a favorable environment for viral reproduction. The HPV E6 proteins utilize the host ubiquitin‐proteosome pathway to target several cellular proteins for degradation. Several HPV proteins are substrates for sumoylation, so the objective of this study was to determine if HPV E6 proteins interacted with or influenced the parallel host sumoylation system. Transient expression of E6 caused an apparent general reduction in the overall level of host sumoylation. To explore the mechanistic basis, we examined the effect of E6 proteins on Ubc9, the sole sumoylation conjugation enzyme. HPV E6 proteins reduced intracellular levels of Ubc9, without affecting transcript levels, so a direct effect on Ubc9 protein stability is likely. Consistent with typical E6‐mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. To examine the effect of E6 on sumoylated proteins in more detail, a fluorescent 2‐dimensional gel system was used to visualize sumoylated proteins. Stable expression of E6 resulted in the loss of 32 sumoylated proteins out of a matched set of over 200 protein spots. Thus, E6 exerts a significant, though selective, effect on host protein sumoylation through its action on Ubc9, which likely helps to create a cellular environment conducive to viral replication. Grant Funding Source : Supported by NIH grant CA089298 to V.G.W.