The Golgi sorting motifs of human cytomegalovirus UL138 are not required for latency maintenance.

Human cytomegalovirus (HCMV) establishes latency within incompletely differentiated cells of the myeloid lineage. The viral protein UL138 participates in establishing and maintaining this latent state. UL138 has multiple functions during latency that include silencing productive phase viral gene transcription and modulating intracellular protein trafficking. Trafficking and subsequent downregulation of the multidrug resistance-associated protein 1 (MRP1) by UL138 is mediated by one of four Golgi sorting motifs within UL138. Here we investigate whether any of the Golgi sorting motifs of UL138 are required for the establishment and/or maintenance of HCMV latency in model cell systems in vitro. We determined that a mutant UL138 protein lacking an acidic cluster dileucine sorting motif unable to downregulate MRP1, as well as another mutant lacking all four Golgi sorting motifs still silenced viral immediate early (IE) gene expression and prevented progeny virion formation during latency. We conclude that the Golgi sorting motifs are not required for latency establishment or maintenance in model cell systems in vitro.

The Membrane-Spanning Peptide and Acidic Cluster Dileucine Sorting Motif of UL138 Are Required To Downregulate MRP1 Drug Transporter Function in Human Cytomegalovirus-Infected Cells.

The human cytomegalovirus (HCMV) UL138 protein downregulates the cell surface expression of the multidrug resistance-associated protein 1 (MRP1) transporter. We examined the genetic requirements within UL138 for MRP1 downregulation. We determined that the acidic cluster dileucine motif is essential for UL138-mediated downregulation of MRP1 steady-state levels and inhibition of MRP1 efflux activity. We also discovered that the naturally occurring UL138 protein isoforms, the full-length long isoform of UL138 and a short isoform missing the N-terminal membrane-spanning domain, have different abilities to inhibit MRP1 function. Cells expressing the long isoform of UL138 show decreased MRP1 steady-state levels and fail to efflux an MRP1 substrate. Cells expressing the short isoform of UL138 also show decreased MRP1 levels, but the magnitude of the decrease is not the same, and they continue to efficiently efflux an MRP1 substrate. Thus, the membrane-spanning domain, while dispensable for a UL138-mediated decrease in MRP1 protein levels, is necessary for a functional inhibition of MRP1 activity. Our work defines the genetic requirements for UL138-mediated MRP1 downregulation and anticipates the possible evolution of viral escape mutants during the use of therapies targeting this function of UL138.IMPORTANCE HCMV UL138 curtails the activity of the MRP1 drug transporter by reducing its steady-state levels, leaving cells susceptible to killing by cytotoxic agents normally exported by MRP1. It has been suggested in the literature that capitalizing on this UL138-induced vulnerability could be a potential antiviral strategy against virally infected cells, particularly those harboring a latent infection during which UL138 is one of the few viral proteins expressed. Therefore, identifying the regions of UL138 required for MRP1 downregulation and predicting genetic variants that may be selected upon UL138-targeted chemotherapy are important ventures. Here we present the first structure-function examination of UL138 activity and determine that its transmembrane domain and acidic cluster dileucine Golgi sorting motif are required for functional MRP1 downregulation.

Human Cytomegalovirus Productively Replicates In Vitro in Undifferentiated Oral Epithelial Cells.

Human cytomegalovirus (HCMV) productive replication in vitro is most often studied in fibroblasts. In vivo, fibroblasts amplify viral titers, but transmission and pathogenesis require the infection of other cell types, most notably epithelial cells. In vitro, the study of HCMV infection of epithelial cells has been almost exclusively restricted to ocular epithelial cells. Here we present oral epithelial cells with relevance for viral interhost transmission as an in vitro model system to study HCMV infection. We discovered that HCMV productively replicates in normal oral keratinocytes (NOKs) and telomerase-immortalized gingival cells (hGETs). Our work introduces oral epithelial cells for the study of HCMV productive infection, drug screening, and vaccine development.IMPORTANCE The ocular epithelial cells currently used to study HCMV infections in vitro have historical significance based upon their role in retinitis, an HCMV disease most often seen in AIDS patients. However, with the successful implementation of highly active antiretroviral therapy (HAART) regimens, the incidence of HCMV retinitis has rapidly declined, and therefore, the relevance of studying ocular epithelial cell HCMV infection has decreased as well. Our introduction here of oral epithelial cells provides two alternative in vitro models for the study of HCMV infection that complement and extend the physiologic relevance of the ocular system currently in use.

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency.

UNLABELLED: The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34(+) hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. IMPORTANCE: Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency but may do so in temporally distinct manners. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs.

An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.