HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14+ Cells

Abstract

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors in vivo. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14+ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14+ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14+ cells.

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+Cell Populations

“…Though their propagation is somewhat laborious, they appear to represent the best compromise between convenience and accuracy. As multiple latency models are now in use (49,50,52,56,67,68), our challenge will be to utilize each to its advantage while integrating results obtained in independent systems into an accurate model for natural HCMV latency.…”

Human Cytomegalovirus Latency and Reactivation in and Beyond the Myeloid Lineage

“…Although the exact roles of the products of these genes during HCMV latency have not been fully established, some insights into their possible roles during latent infection have been determined. For instance, UL138 has been suggested to be important for maintenance of latent infection either as a factor required for latent carriage or as a factor to suppress reactivation -at least in some experimental latent models [49]; the latent viral IL-10 can cause downregulation of MHC class II on the cell surface of cells [51]; LUNA has been reported to be important for reactivation from latency [52]; US28 is a chemokine receptor that responds to CC chemokines and is also constitutively active [53] and UL144 is a TNF family ligand for HVEM [54,55] that shows an ability to indirectly enhance the TH2 immune response in model systems and, thus, may act to suppress the TH1 immune response and avoid immune detection [56].…”

Human Cytomegalovirus Modulates Monocyte-Mediated Innate Immune Responses during Short-Term Experimental LatencyIn Vitro

“…To that end, we analyzed the shortterm outcome of infection of CD14 ϩ peripheral blood monocytes. A shorter time course of infection was chosen to mimic the life span of monocytes in circulation as well as to improve upon the extended culture of monocytes in previous latency models (12,25). Additionally, this time frame would coincide with the established role of the infected monocyte in HCMV dissemination (26,27).…”