Retrovirus-infected Cells Research Articles

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

RUNX1-EVI1 induces dysplastic hematopoiesis and acute leukemia of the megakaryocytic lineage in mice

The RUNX1-EVI1 gene generated by the t(3;21) translocation encodes a chimeric transcription factor and is a causative gene in the development of de novo acute megakaryoblastic leukemia and leukemic transformation of hematopoietic stem cell tumors. Heterozygous RUNX1-EVI1 knock-in mice die in utero due to hemorrhage in the central nervous system and spinal cord and complete abolishment of definitive hematopoiesis in the fetal liver. On the other hand, the chimeric knock-in mouse develops acute megakaryoblastic leukemia. We created another mouse model of RUNX1-EVI1 using transplantation of retrovirus-infected bone marrow cells. Some mice transplanted with RUNX1-EVI1-expressing bone marrow cells developed acute megakaryoblastic leukemia within eight months, and the other non-leukemic mice showed thrombocytosis at around a year. In the non-leukemic mice, dysplastic megakaryocytes proliferated in the bone marrow and frequently infiltrated into the spleen, which was not associated with marrow fibrosis. In the leukemic mice, their tumor cells were positive for c-kit and CD41, and negative for TER119. Although they were negative for platelet peroxidase in the electron microscopic analysis, they had multiple centrioles in the cytoplasm, which are characteristic of megakaryocytes that undergo endomitosis. The leukemic cells were serially transplantable, and gene-expression analyses using quantitative RT-PCR arrays revealed that they showed significantly elevated expression of stem cell, primitive hematopoietic cell and endothelial cell-related genes compared with normal bone marrow cells. All these data suggested that RUNX1-EVI1 caused dysplastic hematopoiesis or leukemia of the megakaryocytic lineage and endowed gene expression profiles distinctive of immature hematopoietic cells.

Exosomes carring gag/env of ALV-J possess negative effect on immunocytes

J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J.

E-102 Exosomes from retrovirus infected cells carry distinct viral noncoding RNAs and proteins that control the fate of the recipient cell

E-102 Exosomes from retrovirus infected cells carry distinct viral noncoding RNAs and proteins that control the fate of the recipient cell

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells.

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.

Correction: PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

The authors would like to correct panel H in Fig 3 to show the correct MFI values of PD-L1 expression on human CD4+ T cells. The error occurred during preparation of the figure for manuscript revision. The percentages of infected CD4 cells expressing PD-L1 were accidentally duplicated from panel I in Fig 3. Please see the corrected version of Fig 3 here. Fig 3 PD-L1 expression on cells infected in vitro with FV or HIV.

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.

Effect of GATA-2 overexpression on function of mouse fetal liver hematopoietic stem cells

This study was aimed to investigate the effect of GATA-2 over-expression on function of mouse fetal liver hematopoietic stem cells. GATA-2 was introduced into mouse fetal liver cells via retrovirus mediated transduction with GFP as a detecting marker. Flow cytometry, colony-forming assay and cell cycle assay were used to detect the biologic changes of these retrovirus infected mouse fetal liver hematopoietic stem cells. The results showed that GATA-2 over-expression increased the Lin(-)Sca1(+)C-Kit(+) (LSK) population dramatically. Cell cycle of LSK cells didn't show abnormal, while colony forming ability decreased significantly. These data indicated that GATA-2 over-expression inhibited definitive differentiation of mouse fetal liver hematopoietic stem cells. It is concluded that over-expression of GATA-2 can significantly raise the LSK cell proportion in mouse fetal liver and inhibit the differentiation capability, the underlying mechanisms may be related to up-regulation of Hes-1, which may lead to the blocking of cell differentiation at the stem/progenitor cell stage.

Transgenic mouse models of the truncated platelet integrin β3 cytoplasmic tail established by stem cell transplantation

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

The 10 Most Read Articles Published in Circulation Research in 2012

The 10 Most Read Articles Published in <i>Circulation Research</i> in 2012

Retrovirus infected cells contain viral microRNAs

The encoding of microRNAs in retroviral genomes has remained a controversial hypothesis despite significant supporting evidence in recent years. A recent publication demonstrating the production of functional miRNAs from the retrovirus bovine leukemia virus adds further credence to the fact that retroviruses do indeed encode their own miRNAs. Here we comment on the importance of this paper to the field, as well as examine the other known examples of miRNAs encoded by RNA viruses.

Human Retrovirus Codon Usage from tRNA Point of View: Therapeutic Insights

The purpose of this study was to investigate the balance between transfer ribonucleic acid (tRNA) supply and demand in retrovirus-infected cells, seeking the best targets for antiretroviral therapy based on the hypothetical tRNA Inhibition Therapy (TRIT). Codon usage and tRNA gene data were retrieved from public databases. Based on logistic principles, a therapeutic score (T-score) was calculated for all sense codons, in each retrovirus-host system. Codons that are critical for viral protein translation, but not as critical for the host, have the highest T-score values. Theoretically, inactivating the cognate tRNA species should imply a severe reduction of the elongation rate during viral mRNA translation. We developed a method to predict tRNA species critical for retroviral protein synthesis. Four of the best TRIT targets in HIV-1 and HIV-2 encode Large Hydrophobic Residues (LHR), which have a central role in protein folding. One of them, codon CUA, is also a TRIT target in both HTLV-1 and HTLV-2. Therefore, a drug designed for inactivating or reducing the cytoplasmatic concentration of tRNA species with anticodon TAG could attenuate significantly both HIV and HTLV protein synthesis rates. Inversely, replacing codons ending in UA by synonymous codons should increase the expression, which is relevant for DNA vaccine design.

Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Abstract 3962 Background:Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods:To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion:Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures:No relevant conflicts of interest to declare.

Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c, and Tbx5

After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

Kinetic Analysis of Porcine Fibroblast Reprogramming Toward Pluripotency by Defined Factors

Induced pluripotent stem cells (iPSCs) are generated from somatic cells through ectopic expression of defined transcription factors. So far, many iPSC lines have been established in various species, including porcine. However, the molecular events during somatic cell reprogramming in pig are largely unknown. The aim of this study was to carry out porcine embryonic fibroblast (PEF) reprogramming by using mouse transcription factors and to monitor morphological and biological progress systematically at an early stage of cell reprogramming. The retrovirus-infected PEF cells retained the four transgenes used and showed morphological changes and alkaline phosphatase staining at day 5 after infection. The endogenous OCT4, NANOG, and TERT genes were activated, and their expression levels were increased significantly. BAX gene expression, a proapoptotic member of the BCL-2 family, was also increased at day 5, suggesting that c-Myc might trigger cell apoptosis. Omission of c-Myc from the cocktail of factors then greatly influenced the reprogramming efficiency and lowered the formation of iPSC colonies. The expression of paternally imprinted DLK1 and DIO3 was slightly downregulated after infection, but then recovered within 2 weeks. However, the expression of maternally imprinted GTL2 was silenced aberrantly at a very early stage of infection and did not recover. Together, these observations illustrated that upregulation of pluripotent-related gene expression, stabilizing the imprinted Dlk1-Dio3 domain, and inhibition of apoptotic events might be conductive to the promotion of the reprogramming process to produce complete porcine iPSCs.

Enforced Pax6 Expression Rescues Alcohol‐Induced Defects of Neuronal Differentiation in Cultures of Human Cortical Progenitor Cells

Alcohol is the most widely consumed substance of abuse, and its use during pregnancy can lead to serious disorders of brain development. The precise molecular action of alcohol on human brain development, however, is still unknown. We previously enriched multipotent progenitor cells, radial glia (RG) cells, from human fetal forebrain and demonstrated that they express transcription factor Pax6 that is necessary for their neurogenic fate. Enriched human fetal RG cells were maintained in vitro as either control or Pax6-expressing retrovirus infected cells. Cultures were treated with increasing doses of alcohol to evaluate Pax6 expression, proliferation, and differentiation of RG cells by immunocytochemistry, Western blot, and RT-PCR methods. In vitro treatment with alcohol reduced the expression of transcription factor Pax6 and proliferation of RG cells, which decreased neurogenesis. Consistent with this finding, the overexpression of Pax6 in RG cells under alcohol treatment rescued cell proliferation and restored the generation of neurons. In contrast to this effect on neurogenesis, the overexpression of Pax6 inhibits the generation of astroglia regardless of alcohol treatment, implying lineage-specific effects. These findings suggest that the effect of alcohol on human neurogenesis is partially due to the reduced expression of transcription factor Pax6 in RG cells.

Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells

Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components.

Natural Killer Cells Recognize Friend Retrovirus-Infected Erythroid Progenitor Cells through NKG2D–RAE-1 Interactions In Vivo

Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

Abstract P1-03-02: Development of a Novel Animal Model of Human Triple-Negative Breast Cancer by Retroviral Oncogene Transduction of Mouse Mammary Stem Cells

Abstract Background: Triple-negative breast cancer (TNBC), which is negative for estrogen receptor (ER), progesterone receptor (PgR), and HER2 expression, represents approximately 20% of all newly diagnosed breast cancers. Patients with TNBC do not derive benefits from endocrine therapy and HER2-targeted therapy. Although TNBC is known to frequently involve mutations of the p53 gene, reduced expression of Rb, and activation of extracellular signal-regulated kinase (ERK), the molecular mechanisms of TNBC's etiology, and carcinogenesis animal models relevant to human TNBC, have not been established. In this study, we developed a mouse model of human TNBC. Methods: To induce TNBC in a murine mammary gland, we used 2 critical genetic events, knockout (KO) of Ink4a/Arf and of the activating form of Hras (12V). Ink4a and Arf play tumor suppressive roles upstream of Rb and p53, respectively; thus, KO of this locus is equivalent to abrogation of the Rb and p53 pathways. Meanwhile, Hras is an oncogene upstream of ERK, Akt, and other kinases. Mammary glands from Ink4a/Arf-KO mice were cultured in serum-free floating culture as single cells, which massed together to form mammospheres. Such mammosphere cells have been shown to repopulate mammary duct-alveolar units in vivo and to differentiate to basal or luminal mammary epithelial cells in adherent culture supplemented with serum, indicating that they are mammary stem cells. To these cellular masses, retroviruses carrying Hras(12V) were infected in floating culture. Then, single-digested mammosphere cells, including the retrovirus-infected cells, were inoculated into mammary fat pads of syngeneic mice. Tumors were evaluated for pathological and immunophenotypical findings and for molecular signature by RT-PCR analysis. Results: Induced tumors showed aggressive features, higher proliferation, dissemination to distant organs, invasiveness to adjacent lesions, and lethality. Pathologically, immature mammary ducts were identified as delineated by positive cells for keratin 14, a marker of basal cells. Immunophenotypically, all induced tumors were negative for ER, PgR, and HER2, although phosphorylation of ERK kinase was strongly and frequently positive. Together, these pathological findings and immune and biological phenotypes are quite similar to those of human TNBC. In addition, the tumors were negative for an epithelial marker, E-cadherin, although the mammospheres were actually derived from mammary epithelial cells. Meanwhile, induced-tumor cells expressed embryonic stem cell-like genes—Oct-3/4, Cripto, and Zfp42, but not Nanog—much more than did Ink4a/Arf-KO or wild-type mammary epithelial cells in RT-PCR analysis. Furthermore, in the induced tumors, we observed heterogeneous expression of CD49f, CD24, and CD44, which are candidate markers of mouse mammary cancer stem cells. Conclusion: Induced tumors had the biological and molecular essence of human TNBC, epithelial-to-mesenchymal transition, dedifferentiation, and cancer stem cells. Therefore, our genetically induced mouse mammary tumor model can help reveal the etiology of this disease from the comprehensive standpoints of pathology, biology, cellular context, and molecular signature. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-02.

CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.