Human Cytomegalovirus Infection Elicits New Decidual Natural Killer Cell Effector Functions

During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56^pos dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues.     

Artesunate Induces Cell Death in Human Cancer Cells via Enhancing Lysosomal Function and Lysosomal Degradation of Ferritin

Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.     

MICA 脱落与肿瘤免疫逃逸：肿瘤免疫治疗新靶标和新策略

MHC Ⅰ类链相关分子（MICA）是自然杀伤细胞和T 细胞上NKG2D 受体的主要活化性配体,在上皮源性肿瘤细胞表面过表达。NKG2D 与MICA 的结合可有效刺激效应细胞对肿瘤细胞的细胞毒作用。然而,临床观察表明,MICA 会在肿瘤的增殖过程中脱落而形成可溶性MICA（sMICA）,这被认为是肿瘤细胞逃脱NKG2D 介导的免疫监视的重要原因。综述在肿瘤细胞中MICA 和NKG2D 的表达与功能、sMICA 的形成与肿瘤免疫逃逸的关联以及介导MICA 脱落的机制,由此探讨肿瘤免疫治疗的新靶点和新策略。     

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.     

Viral Regulation of Cell Tropism in Human Cytomegalovirus

The viral glycoproteins that decorate enveloped viruses play crucial roles in cell entry and in large part dictate the spectrum of cell types that a virus can infect. The identification in human cytomegalovirus (HCMV) of a viral endoplasmic reticulum (ER)-resident glycoprotein that regulates the composition of alternative viral envelope glycoprotein complexes raises the intriguing possibility that certain viruses might actively regulate the tropism of progeny virions to improve their fitness or to navigate through the host.     

The Tiers and Dimensions of Evasion of the Type I Interferon Response by Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a member of the β-herpesvirus family that invariably occupies hosts for life despite a consistent multi-pronged antiviral immune response that targets the infection. This persistence is enabled by the large viral genome that encodes factors conferring a wide assortment of sophisticated, often redundant phenotypes that disable or otherwise manipulate impactful immune effector processes. The type I interferon system represents a first line of host defense against infecting viruses. The physiological reactions induced by secreted interferon act to effectively block replication of a broad spectrum of virus types, including HCMV. As such, the virus must exhibit counteractive mechanisms to these responses that involve their inhibition, tolerance, or re-purposing. The goal of this review is to describe the impact of the type I interferon system on HCMV replication and to showcase the number and diversity of strategies employed by the virus that allow infection of hosts in the presence of interferon-dependent activity.     

Human and Murine Cytomegalovirus Evasion of Cytotoxic T Lymphocyte and Natural Killer Cell-Mediated Immune Responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8⁺T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed.     

Polo-Like Kinase 1 as a Target for Human Cytomegalovirus pp65 Lower Matrix Protein

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.     

Novel Functions for Cell Cycle Genes in Nervous System Development

Many cell cycle genes are known to play important roles in regulating proliferation in the nervous system, however, a growing body of research has proposed that these genes have diverse functions beyond cell cycle regulation. Through the study of new genetic models, cell cycle regulatory genes have been shown to impact on a number of processes during nervous system development including apoptosis, differentiation, and, most recently, neuronal migration. Here we emphasize that the proposed roles for cell cycle genes in neuronal differentiation and migration are not the consequence of deregulated cell cycle, but represent truly novel functions for cell cycle genes.     

Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture

BackgroundHepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.AimTo investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.MethodHCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.ResultsFFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).ConclusionOur study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.     

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-Δm06m152-SIINFEKL, respectively, expressing the K^b-presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ∼10,000 K^b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ∼10% of all cell surface K^b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply.CD8 T cells recognize infected cells by interaction of their T-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77, 85, 97, 98). The number of such “peptide receptors” per cell has been estimated to be on the order of 10⁵ to 10⁶ for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9, 61, 67, 75, 76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-based epitope-specific cell sorting, as well as cells of a peptide-selected cytolytic T-lymphocyte line, protected against mCMV expressing the cognate antigenic peptide, the IE1 peptide 168-YPHFMPTNL-176 in this example, but failed to control infection with a recombinant mCMV expressing a peptide analogue in which the C-terminal MHC-I anchor residue leucine was replaced with alanine (3).Interference with the MHC-I pathway of antigen presentation has evolved as a viral immune evasion mechanism of CMVs and other viruses, mediated by virally encoded proteins that inhibit MHC-I trafficking to the cell surface (for reviews, see references 1, 24, 27, 29, 63, 70, 71, 84, and 95). These molecules are known as immunoevasins (50, 70, 89), as “viral proteins interfering with antigen presentation” (VIPRs) (95), or as negative “viral regulators of antigen presentation” (vRAPs) (34). Although the detailed molecular mechanisms differ between different CMV species in their respective hosts, the common biological outcome is the inhibition of antigen presentation. Accordingly, downmodulation of MHC-I cell surface expression is a hallmark of molecular immune evasion and actually led to the discovery of this class of molecules. Since CD8 T cells apparently protect against infection with wild-type CMV strains despite the expression of immunoevasins, the in vivo relevance of these molecules is an issue of current interest and investigation (for a review, see reference 14). As shown recently with the murine model, antigen presentation in infected host cells is not completely blocked for all epitopes, because pMHC complexes that are constitutively formed in sufficiently large amounts can exhaust the inhibitory capacity of the immunoevasins (40). Likewise, enhancing antigen processing conditionally with gamma interferon (IFN-γ) aids in peptide presentation in the presence of immunoevasins (18, 28). Thus, by raising the threshold of the amount of peptide required for presentation, immunoevasins determine whether a particular viral peptide can function as a protective epitope—an issue of relevance for rational vaccine design as well (94). Whereas deletion of immunoevasin genes gives only incremental improvement to the control of infection in immunocompetent mice (22, 51), expression of immunoevasins reduces the protective effect of adoptively transferred CD8 T cells in immunocompromised recipients (37, 40, 47, 48). In a bone marrow transplantation model, immunoevasins were recently found to contribute to enhanced and prolonged virus replication during hematopoietic reconstitution and, consequently, also to higher latent viral genome loads in the lungs and a higher incidence of virus recurrence (4). Notably, however, immunoevasins do not inhibit but, rather, enhance CD8 T-cell priming (5, 21, 22, 56), due to higher viral replication levels in draining lymph nodes associated with sustained antigen supply for the cross-priming of CD8 T cells by uninfected antigen-presenting cells (5).For mCMV, three molecules are proposed to function as vRAPs, only two of which are confirmed negative regulators that downmodulate cell surface MHC-I (34, 62, 89) and inhibit the presentation of antigenic peptides to CD8 T cells (34, 62). Immunoevasin gp40/m152 transiently interacts with MHC-I molecules and mediates their retention in a cis-Golgi compartment (96), whereas gp48/m06 stably binds to MHC-I molecules in the ER and mediates sorting of the complexes for lysosomal degradation by a mechanism that involves the cellular cargo sorting adaptor proteins AP1-A and AP3-A (73, 74). The third proposed immunoevasin of mCMV, gp34/m04 (46), also binds stably to MHC-I molecules. A function as a CD8 T-cell immunoevasin was predicted from some alleviation of immune evasion for certain epitopes and MHC-I molecules in cells infected with the deletion mutant mCMV-Δm04 (34, 42, 89), but gp34/m04 does not reduce the steady-state level of cell surface class I molecules and does not inhibit peptide presentation when expressed selectively after infection with mCMV-Δm06m152 (34, 62). The m04-MHC-I complexes are expressed on the cell surface (46) and appear to be involved in the modulation of natural killer cell activity (45).Here we give the first report on quantitating the efficacy of immunoevasins in terms of absolute numbers of pMHC complexes displayed at the cell surface. By comparing the fate of pMHC complexes already present at the cell surface in advance of immunoevasin gene expression with that of newly formed pMHC complexes, our data provide direct evidence to conclude that downmodulation of cell surface MHC-I molecules is secondary to an interruption of the flow of newly formed pMHC complexes to the cell surface.(Part of this work was presented at the 12th International CMV/Betaherpesvirus Workshop, 10 to 14 May 2009, Boston, MA.)     

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.     

SLC17A9 Protein Functions as a Lysosomal ATP Transporter and Regulates Cell Viability

Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.     

Role of Type I Interferon Receptor Signaling on NK Cell Development and Functions

Type I interferons (IFN) are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK) cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR) signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27⁺CD11b⁺ double positive (DP) compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.     

Characterization of Two Novel Propachlor Degradation Pathways in Two Species of Soil Bacteria

Propachlor (2-chloro-N-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium, Acinetobacter strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of Pseudomonas (PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain PEM1 grew on propachlor with a generation time of 3.4 h and a K_s of 0.17 ± 0.04 mM. Acinetobacter strain BEM2 grew on propachlor with a generation time of 3.1 h and a K_s of 0.3 ± 0.07 mM. Incubations with strain BEM2 resulted in accumulation of N-isopropylacetanilide, N-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by Pseudomonas sp. strain PEM1 proceeds through a different pathway from metabolism by Acinetobacter sp. strain BEM2.     

Proteomic Screening of Human Targets of Viral microRNAs Reveals Functions Associated with Immune Evasion and Angiogenesis

Kaposi''s sarcoma (KS) is caused by infection with Kaposi''s sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.     

Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.