Reactivation of latent human cytomegalovirus (HCMV) infection following transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance requires expression of UL138. Using SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface Multidrug Resistanceassociated Protein-1 (MRP1), and reduction of substrate export by this transporter. Latencyassociated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14 + and CD34 + progenitors, all in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently-infected cells prior to transplantation.

Many viruses antagonize tumor necrosis factor alpha (TNF-␣) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-␣ receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-␣ has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-␣ responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-␣ on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-␣-mediated reactivation of HCMV.

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms.

NF-B plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-B is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-B in both antiviral defense mechanisms and viral physiology. Here we show that the NF-B signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤) becomes inhibited in HCMV-infected cells. The block to NF-B signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-B-activating pathways, i.e., the IB kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-␣-induced NF-B signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-␣-induced NF-B signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-B signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.