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Characterization of the HCMV protein LUNA and the cellular protein CyPA during latency/reacitvation
AuthorKeyes, Lisa R.
AdvisorSt. Jeor, Stephen C.
Biochemistry and Molecular Biology
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Human cytomegalovirus (HCMV) is a member of the Herpesviridae family. Once an individual is infected with a HCMV, they will harbor the virus for life. As with all herpesviruses, HCMV can yield either a lytic (productive) or latent (non-productive) infection. Latent HCMV can be found in cells of the myeloid lineage, and reactivation from latency occurs when these cells terminally differentiate. Primary HCMV infection is asymptomatic in most individuals. Nevertheless, severe pathology can be associated with HCMV due to viral reactivation from latency. However, the viral and cellular mechanisms that control the switch between lytic and latent infections remain to be determined. For other members of the herpesvirus family, a certain set of genes has been identified for the regulation and maintenance of the latent state of the virus. To date, the specific profile of latent genes for HCMV has not been fully described. An antisense transcript (ast), in the Unique Long (UL) region of the HCMV genome, coding for the UL82 gene; UL81-82ast, has been identified as a potential latency associated transcript. This transcript was isolated from the bone marrow of a healthy HCMV positive individual, in the absence of lytic gene expression, suggesting it may play a role in latent HCMV infections. UL81-82ast codes for a protein known as Latency Unique Natural Antigen (LUNA). Further studies have been carried out to determine the role and importance of this gene in HCMV infections. First, we were able to determine that the LUNA protein is expressed in HCMV seropositive individuals. Standard co-immunoprecipitation and ELISA assays were used to detect antibodies against the LUNA protein in the sera of HCMV seropositive donors. Co-immunoprecipitation for LUNA-specific antibodies was done using a flag-tagged version of purified recombinant LUNA protein expressed in mammalian cells. In order to confirm that our pull downs were specific for antibodies against LUNA, a number of controls were done. As a positive control, flag tag specific antibodies were pulled down using the Flag-tagged LUNA protein. No antibodies were pulled down from human serum using purified flag tag alone, in absence of LUNA. On the other hand we could pull down anti-flag antibodies using this purified flag tag alone. We could only pull down antibodies specific for LUNA protein from HCMV seropositive sera. These results were further confirmed using a standard ELISA to detect antibodies against LUNA. In this case, a His-tagged version of purified recombinant LUNA protein expressed in bacterial cells was used as the antigen. In order to confirm that our ELISA was specific for antibodies against LUNA, a number of controls were done. Specificity was assessed using sera from either normal rabbit sera or from the sera of a rabbit immunized with LUNA protein. Furthermore, of 100 human sera tested, only the 49 HCMV seropositive sera were also positive for presence of specific anti-LUNA antibodies. The likelihood that the perfect degree of correlation observed is due to non-specific interactions is extremely small. These data confirms that LUNA is expressed during in vivo infections and is capable of eliciting an immune response.In order to fully characterize the function of the LUNA protein, we created a LUNA knockout virus (FIX-ΔLUNA) in which the expression of LUNA was inhibited by altering the LUNA start codon, and adding a stop codon 20nt downstream. In the absence of LUNA, we were unable to rescue viable virus from latently infected primary CD14+ monocytes following differentiation. There was a delay in the establishment of latency, as seen by a late switch off of Immediate Early (IE) genes expression in these cells. IE gene expression was not recovered following differentiation of the primary CD14+ monocytes infected with FIX-ΔLUNA. We were also unable to detect UL138 in both human fibroblast cells (HF) and primary CD14+ monocytes infected with FIX-ΔLUNA. Since UL138 has been associated with the establishment of latency, these data suggests that LUNA could be affecting several processes involved in latency and reactivation. Furthermore, we reported that FIX-ΔLUNA produced statistically higher viral copy numbers over time in both HF and primary CD14+ monocytes compared to FIX-WT. We determined that the LUNA protein is essential for the reactivation of HCMV from latently infected CD14+ cells, and that LUNA may play a role in lytic DNA replication.Lastly, to further identify cellular factors involved in HCMV latency/reactivation, we characterized the role of the cellular protein Cyclophilin A (CyPA) in both a typical lytic and latent HCMV infection. CyPA has been demonstrated to play a role during the replication and the production of infectious particles for a number of different viruses, including murine cytomegalovirus (MCMV). In this study, we used HF cells to model our lytic infection, and monocyte-like THP-1 cells as a model for a latent HCMV infection. Infection of THP-1 cells is an established model commonly used to study HCMV latency and reactivation. It was previously demonstrated that upon differentiation using Phorbol 12-myristate 13-acetate (PMA), HCMV could be rescued from experimental latency using a mechanism that closely mimics infection of primary myeloid cells. We characterized the effects of CyPA on both a lytic and latent infection by either adding in exogenous CyPA, or silencing CyPA with siRNA. We did not observe any significant changes in the production of infectious virions in the presence of exogenous CyPA. When we silenced CyPA, we observed a significant decrease in viral DNA loads in infected HF cells over time, suggesting that CyPA may be involved in lytic DNA replication. Interestingly, when CyPA was silenced, we were unable to recover infectious virus following differentiation of infected THP-1 cells. Taken together, these data suggests that CyPA is involved in lytic DNA replication, and that CyPA is a necessary factor involved in HCMV reactivation in latently infected THP-1 cells.