Pomalidomide restores immune recognition of primary effusion lymphoma through upregulation of ICAM-1 and B7-2.

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi's sarcoma, a tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom's effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom's interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.

Human Cytomegalovirus Interactions with the Basement Membrane Protein Nidogen 1.

In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus.IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

Human Cytomegalovirus Compromises Development of Cerebral Organoids.

Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker ß-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects.IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker ß-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.

Mechanism and therapeutic implications of pomalidomide-induced immune surface marker upregulation in EBV-positive lymphomas.

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/ß in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom's effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.