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Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality among both solid organ and hematopoietic stem cell transplant recipients. Identification of cells throughout the body that can potentially serve as a viral reservoir is essential to dissect mechanisms of cell tropism and latency and to develop novel therapies. Here, we tested and compared the permissivity of liver-, brain-, lung (LNG)- and bone marrow (BM)-derived perivascular mesenchymal stromal cells (MSC) to HCMV infection and their ability to propagate and produce infectious virus. Perivascular MSC isolated from the different organs have in common the expression of CD146 and Stro-1. While all these cells were permissive to HCMV infection, the highest rate of HCMV infection was seen with LNG-MSC, as determined by viral copy number and production of viral particles by these cells. In addition, we showed that, although the supernatants from each of the HCMV-infected cultures contained infectious virus, the viral copy number and the quantity and timing of virus production varied among the various organ-specific MSC. Furthermore, using quantitative polymerase chain reaction, we were able to detect HCMV DNA in BM-MSC isolated from 7 out of 19 healthy, HCMV-seropositive adults, suggesting that BM-derived perivascular stromal cells may constitute an unrecognized natural HCMV reservoir.

Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.

Human cytomegalovirus (HCMV) latency is poorly understood. We previously described a novel HCMV latency-associated transcript, UL81-82ast, coding for a protein designated LUNA (latency unique natural antigen). The aim of this study was to confirm the presence of LUNA in HCMV-seropositive donors. Standard co-immunoprecipitation and ELISA assays were used to detect antibodies against the LUNA protein in the sera of HCMV-seropositive donors. Specific antibodies against LUNA were detected in all HCMV-seropositive donors but in none of the seronegative donors. These data confirm that LUNA is expressed during in vivo infections and is capable of eliciting an immune response.

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science , this issue p. 755 ; see also p. 700

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.

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells., Author Summary Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma herpesvirus are widespread pathogens that establish lifelong infections in a dormant state termed latency. Although most gammaherpesvirus infections are asymptomatic, infection of some individuals leads to the development of B cell lymphoma or other cancers. It is well known that during latency these viruses reside in mature B cells of the immune system; however, little is known about how this reservoir is maintained for life. Using murine gammaherpesvirus 68 infection of mice as a model to study gammaherpesvirus infections inside a living host, we have previously demonstrated that gammaherpesviruses can infect early precursors of B cells. In normal situations, the differentiation of such precursors into mature B cells is a tightly regulated process that leads to the death of cells that react inappropriately to host tissues. Here though, we demonstrate that a gammaherpesvirus protein called vBcl-2 can block the death of infected precursor B cells, and that vBcl-2 is critical for infection of these cells. Finally, we show that depleting precursor B cells reduces mature B cell latency. Together, these data suggest that vBcl-2 proteins play a key role in lifelong gammaherpesvirus latency and may be a potent target for future drug development.

Abstract 3464 Cytomegalovirus (HCMV) infection or reactivation is an important cause of morbidity and mortality among hematopoietic cell transplantation (HCT) recipients. HCMV infection in these patients may result from a new HCMV infection, reactivation of pre-existing latent virus, or both. Regardless, HCMV causes end organ diseases including pneumonia, gastroenteritis, retinitis, hepatitis, and encephalitis, and can contribute to graft failure. Although the use of antiviral drugs has decreased the incidence, morbidity, and mortality of HCMV disease in transplant recipients, identification of cells serving as the reservoir and/or that are responsible for reactivation of the disease could potentially lead to the development of novel targeted therapies. Although hematopoietic cells, particularly myeloid progenitors, have been identified as reservoirs for HCMV reactivation, it is possible that within the bone marrow environment there are cells that are able to serve as a reservoir for this virus. Perivascular mesenchymal/stromal cells (MSC) are part of the microenvironmental niche and play a role in hematopoietic stem cell (HSC) maintenance and differentiation. Because MSC have an increased resistance to chemo- and radio-therapy, allowing the survival of resident MSC during HCT, they express CD29, a main receptor for HCMV capture and entry, and they directly interact with HSC within the niche, we hypothesized that MSC could serve as a potential reservoir and a source of HCMV reactivation in transplantation patients. To this end, we collected bone marrow (BM) from 10 different healthy adult donors, who had tested positive for CMV antibodies, and investigated whether MSC (phenotypically defined as cells positive for CXCL12, CD90, CD44, CD29, CD73, CD105, and CD146, but negative for both CD34 and CD45) were positive for CMV, by testing for the presence of Immediate Early (IE) HCMV gene products by PCR with subsequent confirmation by sequencing. MSC sorted from 7 out of 10 adult donors tested positive for the presence of IE HCMV DNA, demonstrating persistence of the virus in these cells in the absence of disease. In order to further evaluate the permissivity of these cells to HCMV infection and to determine whether the virus was able to establish a lytic infection, we infected MSC from HCMV-negative donors in vitro, with the clinical isolate Davis strain of HCMV (MOI of 3). At 1, 3, 5, and 7 days post-infection (dpi), the cells were positive for IE HCMV DNA. They were also positive, at one or more of the tested time points, for UL-83, a late gene associated with lytic infection. IE protein expression was also detected by immunohistochemistry. The mock infected cells remained negative for both IE and UL-83 throughout the duration of the experiment. In order to determine whether MSC were able to produce and release infectious viral particles, supernatants were collected at different times post-infection, and evaluated for the presence of infectious units (IU) using HFF. At 1, 3, 5, and 7 dpi, MSC produced 5167±809, 6867±104, 3800±200, and 3467±788 IU, respectively, demonstrating that MSC are able to produce and release infectious particle and infect neighboring cells. We have therefore shown that a highly defined population of human stromal cells (MSC) naturally harbors latent HCMV. Since MSC reside within/comprise the HSC niche, they share an intimate relationship with hematopoietic progenitors, and they participate in homing and retention of HSC, our data support the conclusion that MSC may serve as a previously unrecognized reservoir for this clinically important virus in transplant recipients. Moreover, upon infection with HCMV, MSC are able to produce infectious virus, thereby propagating HCMV infection to adjacent cells. Studies are underway to determine whether productive HCMV infection of this stromal cell population also alters the expression of proteins that regulate HSC maintenance. Disclosures: No relevant conflicts of interest to declare.