Functional characterization of the US16 gene of Human Cytomegalovirus

The Human Cytomegalovirus (HCMV) represents the major viral cause of birth defects, as well as an important opportunistic pathogen for immunocompromised individuals. The functional profiling of the whole HCMV genome in a bacterial artificial chromosome (BAC)-based clone derived from a laboratory strain of the virus, allowed to identify those viral genes which are essential or dispensable for productive replication in the standard host cell model represented by primary fibroblasts. These HCMV dispensable genes include also those which regulate one the most intriguing and for a long time contradictory feature of HCMV biology, that is the exceptionally wide range of cell types in which the virus can enters, expresses its genes and productively replicates. In fact, in spite of its strict host specificity, the virus can spread virtually to any organ system during acute infections and under certain conditions such as severe cases of congenital infections. The long list of susceptible cells include some of the predominant targets for in vivo virus replication, such as epithelial cells of glands and mucosal tissues, fibroblasts of connective tissues, smooth muscle cells in the gastrointestinal tract and vascular endothelial cells. Among the HCMV non¿essential genes for in vitro replication in fibroblasts, the US12 gene family contains a series of ten tandemly arranged genes from US12 until US21 located in the unique short (US) region of the genome. The aim of this work was to investigate the pattern of expression and the functional relevance of the US16 gene, a member of the US12 gene family, in the context of the replication of a clinical isolate of HCMV (TR) in different cell types. To this purpose, we firstly tagged the US16 ORF with a HA epitope tag at its carboxyl terminus in a recombinant virus derived from the TR strain. Immunoblot analysis showed the expression of a single protein band with an apparent molecular mass of about 33 kDa starting from 48 h.p.i. with true late (L) gene kinetic. In infected cells, pUS16 localized in the cytoplasmic virus assembly compartment (VAC) together with gB and pp28 proteins, but, unlike these two viral late proteins, pUS16 is not detected in purified extracellular virions. The generation of pUS16-deficient viruses from the clinical isolate TR then allowed to examine the contribution of pUS16 to HCMV replication in different cell types. In endothelial, epithelial and dendritic cells, US16-null viruses exhibited a severe growth defect that occurs prior to the expression of IE proteins, thus indicating that pUS16 is needed for efficient infection and productive replication in these cell types. A partial rescue of the US16- deficient virus infectivity was observed with the virus progeny released from the producer fibroblast cells that had been transduced with an adenoviral vector expressing an US16-V5 protein. Together, these results demonstrated that pUS16 is required for the efficient infection in endothelial, epithelial and dendritic cells, and that pUS16 may mediate a modification of virus particles in the producer cells that confers them the competence for infection of these cell types. Hence, the results of this work led to the conclusion that the US16 gene of HCMV encodes a new viral tropism factor required for a productive replication in least three different cell types that are known to be important for HCMV replication, dissemination, and persistence in the natural host.