The role of UL128 and RL13 in cell-associated spread of human cytomegalovirus

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Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent in 40-100 % of the population worldwide and causes significant morbidity under conditions of reduced immune defenses. In principle, HCMV can spread both cell-free by releasing infectious virions into the extracellular space and cell-associated by direct cell-to-cell transmission of viral progeny. Apparently, dissemination within that host is greatly due to the cell-associated mode. Infectivity in the bloodstream is found almost exclusively in the leukocyte fraction, and freshly isolated HCMV almost always spreads in a strictly cell-associated manner.The general objective of this project is to make substantial progress in the investigation of the molecular factors underlying cell-associated spread of HCMV, as we are convinced that this ill-defined transmission mode is a key for better control of the virus in clinically relevant situations. The viral genes RL13 and UL128 were identified as factors that keep HCMV cell-associated, but their mode of action is largely unknown, since they are disrupted quickly after isolation of HCMV from clinical specimens. Loss of RL13 and UL128 provides a strong selective advantage by promoting cell-free spread. This why intact viral genomes have not successfully been cloned in bacterial vectors (BACs), which is a prerequisite for genetic engineering for detailed molecular studies.We aim to overcome such limitations and to gain substantive insights regarding the detailed contribution of viral and cellular factors involved in cell-associated spread of HCMV.1. Technically, we address cloning of recent HCMV isolates as BACs, which would allow for the first time the targeted genetic modification of such isolates. We have developed a novel method for releasing cell-free infectivity from isolate-infected fibroblast cultures that opens this option. We will generate a collection of BAC-cloned HCMV variants representing all genotypes of the various polymorphic glycoproteins, and we will construct reporter viruses suitable for antiviral screening approaches or live imaging of virus particles. These collections of viruses will be shared with the scientific community2. Concerning the molecular mechanisms underlying cell-associated spread, we will focus on the role of the viral genes UL128 and RL13. We will investigate the dose-response relationship between their expression and cell-association of the virus and analyze whether the two proteins interact in infected cells and how they reduce cell-free infectivity.In an attempt to develop culture models reflecting virus shedding compartments, we will try to identify cell types or culture conditions that allow the release of cell-free virus from isolates e.g. by downmodulation of UL128 or RL13.Taken together, the intended advances in this project are meant to provide a basis for the future development of antiviral agents that specifically target the cell-associated spread of HCMV.