Inhibition of Human Peptide Deformylase Disrupts Mitochondrial Function

Deformylases are metalloproteases in bacteria, plants, and humans that remove the N-formyl-methionine off peptides in vitro. The human homolog of peptide deformylase (HsPDF) resides in the mitochondria, along with its putative formylated substrates; however, the cellular function of HsPDF remains elusive. Here we report on the function of HsPDF in mitochondrial translation and oxidative phosphorylation complex biogenesis. Functional HsPDF appears to be necessary for the accumulation of mitochondrial DNA-encoded proteins and assembly of new respiratory complexes containing these proteins. Consequently, inhibition of HsPDF reduces respiratory function and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. A series of structurally different HsPDF inhibitors and control peptidase inhibitors confirmed that inhibition of HsPDF decreases mtDNA-encoded protein accumulation. Therefore, HsPDF appears to have a role in maintenance of mitochondrial respiratory function, and this function is analogous to that of chloroplast PDF.The human mitochondrial protein peptide deformylase, HsPDF, is a metalloprotease that removes the formyl moiety on the methionine of N-formyl-methionine peptide substrates in an enzymatic assay (24, 35). Despite the slow kinetic properties of HsPDF in an in vitro deformylation assay (24, 29, 35), we have shown that small interfering RNA (siRNA) interference of HsPDF decreases human cancer cell proliferation. Similarly, pharmacologic inhibition with the PDF antibiotic inhibitor actinonin and its analogs results in mitochondrial membrane depolarization and promotes cell death or proliferation arrest in a wide variety of cancer cell lines (18, 25). However, the cellular function of HsPDF remains elusive, and others have proposed that it has none (29). In bacteria, deformylation of nascent peptides is necessary for removal of the N-terminal methionine (36) and posttranslational processing of at least a subset of proteins that contribute to cell growth and viability (28). Prokaryotic PDF thus fulfills a role in cotranslational processing (7) and in protein degradation (41).In mammals, N-terminal formylation of proteins is only known to occur during mitochondrial translation initiation, as in prokaryotic protein translation (6). In contrast to bacteria, where the entire proteome is formylated for translation initiation, formylation in eukaryotes is limited to the 13 mitochondrial DNA (mtDNA)-encoded proteins. Formylation is important for mitochondrial translation, because formyl-Met-tRNA, but not Met-tRNA, is recognized by initiation factor 2 as the initiator tRNA (26, 37, 39). Therefore, the participation of HsPDF in protein post- or cotranslational processing can be narrowed down to these mitochondrial translation products.Despite the current understanding of the function of formyl-methionine in the initiation of protein synthesis in mammalian mitochondria (38, 39), the functional relevance of the downstream processing of nascent mitochondrial translation products has remained unexplored. Furthermore, it has been assumed that human mitochondria-encoded proteins, like those of bovine origin, are generally not deformylated after synthesis (45).The mammalian mitochondrial genome-encoded proteins are all subunits of four of the five oxidative phosphorylation respiratory chain enzyme complexes (I, III, IV, and V) (2, 40, 42). Respiratory complexes are comprised of multiple proteins. With the exception of complex II, which is comprised entirely of nuclear DNA-encoded subunits, all other complexes include both nuclear and mitochondrial DNA-encoded proteins. Synthesis of key mtDNA-encoded protein subunits, and the assembly of these proteins with multiple nuclear-encoded subunits within the mitochondria, is necessary for the function of each individual complex (16, 30, 44). Moreover, a functional interdependence among stably assembled respiratory complexes has been demonstrated (1). Mutations in human mtDNA that affect protein-coding regions or nuclear DNA mutations that affect expression of respiratory complex subunits cause disease (13), including Parkinson''s disease, for example, in which decreased respiratory function and compromised cell viability have been demonstrated (5, 21, 23). Therefore, the importance of properly assembled mitochondrial respiratory complexes suggests that their disruption, by inhibition of mtDNA-encoded protein processing, could have significant effects on cellular function.We hypothesized that HsPDF-mediated processing of mtDNA-encoded proteins is necessary for proper function of the respiratory chain complexes. To determine how the human deformylase activity contributes to cellular function, we used pharmacologic inhibition of HsPDF activity with the hydroxamic acid peptidomimetic inhibitor of PDF, actinonin, and confirmed our findings with a variety of other structurally different inhibitors. PDF has been shown to be a target of actinonin in bacteria (9), human cells (24), and plants (17).Here we show that inhibition of HsPDF function in mitochondria of human cell lines reduces mtDNA-encoded protein accumulation, new respiratory complex assembly, and energy production by the mitochondria. Aerobic glycolysis-dependent cell survival ensues upon disruption of HsPDF function. Therefore, HsPDF appears to fulfill a function in the mitochondria and to have a role in mtDNA-encoded protein-containing oxidative phosphorylation (OXPHOS) complex biogenesis.     

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.     

Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.DNA damage arises from errors in the replication process, as well as a myriad of intrinsic and extrinsic DNA-damaging agents that continually assault cells. Failure to efficiently repair DNA lesions leads to accumulation of mutations that contribute to numerous pathologies, including carcinogenesis. In addition to genomic DNA, mitochondrial DNA (mtDNA) is subject to damage that requires repair to maintain integrity. For these reasons, it is not surprising that DNA replication and repair proteins display significant plasticity that allows participation in several divergent replication and repair processes. In addition, numerous mechanisms, including alternative splicing, posttranslational modifications, or utilization of alternative translation initiation start sites, allow DNA replication and repair proteins such as Pif1, DNA ligase III, and APE1 to localize to the nucleus and the mitochondrion and participate in DNA replication and/or repair (9, 17, 25), thus ensuring genomic DNA and mtDNA integrity.Dna2 is an evolutionarily conserved helicase/nuclease enzyme. Originally discovered in Saccharomyces cerevisiae, Dna2 orthologs are found throughout the animal kingdom, including humans (5, 22, 28). Early studies demonstrated that Dna2 functions in concert with Flap endonuclease 1 (FEN1) to remove long DNA flaps that form upon lagging-strand DNA replication (6). However, in contrast to FEN1, Dna2 is an essential gene in yeast, suggesting that other proteins, including FEN1, cannot compensate for its loss in DNA replication or that it possesses functions beyond its role in Okazaki fragment processing. In agreement with this, genetic and biochemical studies have implicated Dna2 in DNA double-strand break (DSB) repair, telomere regulation, and mitochondrial function (8, 10, 15, 26, 38, 44, 45).Analysis of Dna2 in yeast revealed that it undergoes dynamic cell cycle localization. Dna2 localizes to telomeres during G₁, relocalizes throughout the genome in S phase, and moves back to the telomere during late S/G₂, where it participates in telomere replication and telomerase-dependent telomere elongation (10). Dna2 also leaves the telomere following treatment with bleomycin and localizes to sites of DNA DSBs (10). In addition, dna2 mutants are sensitive to DNA damage induced by gamma radiation and methanesulfonic acid methyl ester (7, 15). These phenotypes may be explained by recent work demonstrating that Dna2 plays an important role in 5′-end resection following DSBs. Indeed, upon induction of DSBs and initiation of 5′-end resection by the Mre11-Rad50-Xrs2 complex, Dna2 and Sgs1 cooperate to further degrade the 5′ end, creating long 3′ strands essential for homologous recombination (26, 45). Finally, while dna2Δ mutations are lethal in budding yeast, the dna2Δ pif1-m2 (nuclear PIF1) double mutations rescue dna2Δ lethality but produce a petite phenotype, suggesting that Dna2 is also involved in mtDNA maintenance (8).Recently, the human ortholog of Dna2 was cloned and characterized (23, 29). Biochemical analysis revealed that, similar to its yeast counterpart, the human Dna2 (hDna2) protein possesses nuclease, ATPase, and limited helicase activities (23, 29), suggesting that it carries out analogous functions in yeast and mammalian cells. However, hDna2''s putative role in genomic DNA repair and replication was called into question by a recent study suggesting that hDna2 is absent from the nucleus and found exclusively within the mitochondria, where it participates in mtDNA repair (44). Further in vitro biochemical studies suggested that hDna2 also participates in mtDNA replication (44). Here, we confirm that hDna2 localizes to the mitochondria and demonstrate that hDna2 participates in mtDNA replication and repair. However, our studies go further by uncovering a nuclear form of hDna2 that plays an important role in genomic stability. Indeed, we demonstrate that depletion of hDna2 leads to the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that hDna2, like its yeast counterpart, is essential to maintain nuclear DNA stability.     

Mitochondrial Dysfunction in Lyssavirus-Induced Apoptosis

Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.During coevolution with their hosts, viruses have developed many ways of manipulating the cellular machinery of infected cells. They inhibit or induce apoptosis for their own benefit, with the purpose of increasing viral replication and spread or subverting the host''s immune response (4, 12, 51, 59).Mitochondria have several functions in the cell, including energy production, calcium buffering, and regulation of cellular apoptosis. Death signals in the intrinsic pathway of apoptosis act directly on mitochondria, leading to their dysfunction and the release of proapoptotic factors responsible for the caspase-dependent and/or -independent death pathways (43). The process is tightly regulated positively or negatively by proteins from the Bcl-2 family (32). Caspase activation can be initiated in the extrinsic pathway of apoptosis by death receptors expressed at the cell surface; this later causes mitochondrial dysfunction (8, 20).Lyssaviruses are highly neurotropic viruses associated with rabies, a fatal encephalomyelitis considered to be a reemerging zoonosis throughout most of the world (10). It has been suggested that lyssavirus-induced neuronal apoptosis (1), previously thought to be a principal cause of pathogenesis, is an important defense mechanism against lyssavirus infection (26, 34, 56). However, the molecular basis of lyssavirus-induced neuronal apoptosis is still poorly understood (16, 55). The involvement of the viral glycoprotein (G) in inducing neuronal apoptosis has been extensively shown (13, 38, 39, 45), whereas we have suggested that M is an inducer of neuronal cell death through a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent pathway (29). However, the molecular mechanism of apoptosis has not been precisely defined, and little is known about mitochondrial involvement during lyssavirus infections (46).In this study, we take advantage of the fact that Mokola virus (MOK), a member of the genotype 3 lyssaviruses (5), is known to be less pathogenic than viruses of genotype 1 and, in particular, Thailand virus (THA) (3). We report for the first time the involvement of the mitochondrial machinery during MOK-induced apoptosis. We show that the MOK matrix protein (M-MOK), a previously described apoptogenic factor (29), interacts directly with cytochrome c (cyt-c) oxidase (CcO) subunit I (CcO1), the terminal component of the mitochondrial respiratory chain (MRC). This finding is of interest, as this interaction, which is not found with M-THA, may have a key role in controlling ATP synthesis and cellular respiration during lyssavirus-induced neuronal apoptosis and may contribute to the low pathogenesis of MOK infection.     

Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.All members of the Herpesviridae encode a conserved alkaline DNase that displays limited homology to bacteriophage λ red α (2, 24), an exonuclease that acts in conjunction with the synaptase red β to catalyze homologous recombination between DNA molecules (23). The most thoroughly characterized member of the herpesvirus alkaline nuclease family is encoded by the herpes simplex virus type 1 (HSV-1) gene UL12 (7, 9, 22). HSV-1 UL12 has both endo- and exonuclease activity (15-17, 36) and binds the viral single-stranded DNA binding protein ICP8 (37, 39) to form a recombinase that displays in vitro strand exchange activity similar to that for red α/β (27). UL12 localizes to the nucleus (26) where it plays an important, but as-of-yet ill-defined, role in promoting the production of mature packaged unit-length linear progeny viral DNA molecules (12, 20), perhaps via a recombination mechanism (27, 28). The importance of UL12 is documented by the observation that UL12 null mutants display a ca. 1,000-fold reduction in the production of infectious progeny virions (41).HSV-1 also produces an amino-terminally truncated UL12-related protein termed UL12.5, which is specified by a separately promoted mRNA that initiates within UL12 coding sequences (7, 9, 21). UL12.5 is translated in the same reading frame as UL12 but initiates at UL12 codon 127 and therefore lacks the first 126 amino acid residues of the full-length protein. UL12.5 retains the nuclease and ICP8 binding activities of UL12 (4, 14, 26) but does not accumulate to high levels in the nucleus (26) and is unable to efficiently substitute for UL12 in promoting viral genome maturation (14, 21). We recently showed that UL12.5 localizes predominantly to mitochondria, where it triggers massive degradation of the host mitochondrial genome early during HSV infection (31). Mammalian mitochondrial DNA (mt DNA) is a 16.5-kb double-stranded circle located within the mitochondrial matrix that encodes 13 proteins involved in oxidative phosphorylation and the RNA components of the mitochondrial translational apparatus (reviewed in reference 10). Inherited mutations that inactivate or deplete mt DNA impair oxidative phosphorylation, leading to a wide range of pathological conditions, including neuropathy and myopathy (reviewed in references 8 and 40). Thus, although the contribution of mt DNA depletion to the biology of HSV infection has yet to be determined, it likely has a major negative impact on host cell functions.UL12 and UL12.5 provide a striking example of a pair of highly related proteins that share a common biochemical activity yet differ markedly in subcellular location and biological function. The basis for their distinct subcellular localization patterns is of considerable interest, as the only difference in the primary sequences is that UL12.5 lacks the first 126 residues of UL12. Reuven et al. (26) demonstrated that this UL12-specific region contains one or more signals able to target enhanced green fluorescent protein (eGFP) to the nucleus. However, the determinants of the mitochondrial localization of UL12.5 have not been previously examined. Most proteins that are imported into the mitochondrial matrix bear a matrix targeting sequence that is located at or close to the amino terminus (reviewed in references 25 and 38). We speculated that UL12.5 bears such an amino-terminal matrix targeting sequence and that the function of this element is masked in the full-length UL12 protein by the UL12-specific amino-terminal extension, which contains the nuclear localization signal(s) (NLS). Our results broadly support this hypothesis and indicate that the mitochondrial localization sequence of UL12.5 is located ca. 60 residues from its N terminus.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via Maintenance of Oxidative Phosphorylation

p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.Tumors can be distinguished from their nonmalignant counterparts by specific molecular signatures expressed in malignant cells and tumor vasculature. We explore such differences by identifying tumor-homing peptides from phage libraries that we screen in vivo (60). We recently showed (19) that the cellular receptor for one of our tumor-homing peptides is a protein variously known as p32, p33, gC1q receptor (gC1qR), or hyaluronic acid binding protein 1 (HABP1). This protein was originally isolated based on its copurification with the nuclear splicing factor SF-2 (37). However, it was subsequently shown to bind also the globular heads of complement component C1q (23), hyaluronic acid (10), and numerous other extracellular and intracellular proteins (24, 28, 33, 42). Most recently it has been shown that p32 interacts with the long and short forms of the tumor suppressor ARF (30, 56, 57). Despite the numerous reports on p32 interaction partners, the role of these binding activities in the physiological function of the protein is unknown, and some investigators have proposed that p32 may be a chaperone protein (58, 65).The p32 protein is primarily localized in the mitochondrial matrix (12, 46, 48) but has also been reported to be present in other subcellular locations (53). Some of the p32 protein can be at the cell surface, a location that appears to be specific for tumors (19). In this regard, p32 is similar to some other intracellular proteins that are also partially localized at the cell surface in tumor cells (8, 49). In addition to the partial cell surface localization of p32, many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues (7, 19, 52, 59). Moreover, p32 is differentially expressed during the progression of epidermal carcinoma, accumulating in metastatic islands (25).We set out to modulate p32 expression in tumor cells to gain information on the role of this protein in cancer. We show here that p32 knockdown cells shift their metabolism from oxidative phosphorylation (OXPHOS) toward glycolysis and become poorly tumorigenic. These changes could be reversed by restored expression of p32. These results show that p32 supports oxidative phosphorylation in human cancer cells and opposes the shift of tumor cell metabolism toward glycolysis. The unique expression pattern of p32 in tumors and its crucial role in tumor metabolism make p32 a promising target for tumor therapy. The fact that this protein is upregulated in tumors and counteracts glycolytic metabolism suggests that the role of the Warburg effect in tumor growth may not be as straightforward as is generally thought.     

Downregulation of Mitochondrial Porin Inhibits Cell Growth and Alters Respiratory Phenotype in Trypanosoma brucei

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a β-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.Trypanosoma brucei belongs to a group of parasitic protozoa that possess a single tubular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast (30). T. brucei is the infectious agent of the disease African trypanosomiasis, which is spread from one mammal to another by the bite of the tsetse fly (53). During transmission from the insect vector to the mammalian host and vice versa, the parasite undergoes various developmental stages accompanied by dramatic changes in mitochondrial activities (15). The bloodstream form that grows in mammalian blood uses glucose as its energy source and suppresses many mitochondrial activities. The bloodstream-form mitochondria lack cytochromes; thus, respiration in this form is solely dependent on the cytochrome-independent trypanosome alternative oxidase (TAO) (15). In contrast, the procyclic form that lives in the insect''s midgut possesses a developed mitochondrion with a full complement of the cytochrome-dependent respiratory system and a reduced level of TAO. The procyclic-form mitochondria produce ATP by both oxidative and substrate-level phosphorylations (7). On the other hand, the bloodstream-form mitochondria do not produce ATP but hydrolyze ATP to maintain the inner membrane (IM) potential (10, 33, 39, 48). Many of the mitochondrial IM- and matrix-localized proteins in T. brucei are well characterized (11, 29, 34, 43, 45). However, the mitochondrial outer membrane (OM) proteins in this group of parasitic protozoa have been poorly explored.Mitochondrial porin, which is also known as the voltage-dependent anion-selective channel (VDAC), is the most abundant protein in the OM (17, 28). The sizes and the secondary structures of this protein are very similar among different organisms. The VDAC possesses a N-terminal α-helical domain, and the rest of the protein consists of a number of amphiphilic β-strands, which form a barrel-like structure that integrates into the lipid bilayer (16, 17, 28). Recently, the three-dimensional structure of the human VDAC has been elucidated by nuclear magnetic resonance spectroscopy and X-ray crystallography, which showed a β-barrel architecture composed of 19 β-strands and the N-terminal α-helix located horizontally midway in the pore (5). Saccharomyces cerevisiae and Neurospora crassa VDACs also possess 16 to 19 β-strands, similar to the mammalian VDAC (17).The VDAC exists as different isomeric forms in different species (16, 19). In yeasts, there are two forms: VDAC1 and VDAC2. Only VDAC1 has the channel activity and is abundantly expressed (22, 23). Animals have three isoforms: VDAC1 to VDAC3. These isoforms showed more than 80% sequence homology among themselves. However, their expression levels and tissue specificities are different (16). Plants also have multiple isoforms of the VDAC with various expression levels under different pathological conditions (19). The VDAC plays a crucial role in regulated transport of ADP, ATP, Ca²⁺, and other metabolites in and out of mitochondria (17, 28, 41). Two ATP-binding sites found at the N- and C-terminal regions in the VDAC are critical for its function (54). Downregulation of VDAC expression disrupts mitochondrial energy production (22, 25). In contrast, overexpression of the VDAC in metazoa induces apoptosis, which can be blocked by compounds that inhibit its channel activity (1, 47).The OM of gram-negative bacteria also consists of various types of porins (24, 32, 40). Based on their structures and functions, they are divided into five groups. OmpA belongs to the small β-barrel integral membrane protein family, which is composed of eight β-strands. It is highly abundant and ubiquitous among most gram-negative bacteria (21). Other types of porins include general porin OmpF, which consists of 16 β-strands; substrate-specific porins, such as LamB or maltoporin, which contains 18 β-strands; receptor-type porin FhuA, the largest β-barrel, with 22 β-strands; and phospholipase A or OMPLA, an integral membrane enzyme containing 12 β-strands (21, 24, 32, 40). The OmpA plays important roles in bacterial conjugation, adhesion, invasion, and immune evasion and also acts as the receptor for several bacteriophages through its surface-exposed loops (44).Here, we show that the T. brucei mitochondrial porin (TbPorin) possesses a predicted β-barrel structure that has fewer β-strands than other mitochondrial porins but is similar to bacterial OmpA. TbPorin is crucial for mitochondrial energy production via both oxidative and substrate-level phosphorylations. The depletion of TbPorin reduced cell growth of the procyclic form as well as the bloodstream form. Furthermore, it reveals that depletion of mitochondrial ATP level by downregulation of porin alters the electron flow via TAO and the cytochrome-dependent pathway (CP) as well as the levels of proteins in these pathways.     

Mitochondrial Dysfunction Contributes to Oncogene-Induced Senescence

The expression of oncogenic ras in normal human cells quickly induces an aberrant proliferation response that later is curtailed by a cell cycle arrest known as cellular senescence. Here, we show that cells expressing oncogenic ras display an increase in the mitochondrial mass, the mitochondrial DNA, and the mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest. By the time the cells entered senescence, dysfunctional mitochondria accumulated around the nucleus. The mitochondrial dysfunction was accompanied by oxidative DNA damage, a drop in ATP levels, and the activation of AMPK. The increase in mitochondrial mass and ROS in response to oncogenic ras depended on intact p53 and Rb tumor suppression pathways. In addition, direct interference with mitochondrial functions by inhibiting the expression of the Rieske iron sulfur protein of complex III or the use of pharmacological inhibitors of the electron transport chain and oxidative phosphorylation was sufficient to trigger senescence. Taking these results together, this work suggests that mitochondrial dysfunction is an effector pathway of oncogene-induced senescence.Mitochondria are central to cell metabolism and energy production. High-energy electrons coming from the oxidation of different carbon sources such as glucose and fatty acids enter the mitochondrial electron transport chain as reduced equivalents, and their energy gradually is converted into a proton gradient. Mitochondria use this gradient to synthesize ATP that later is used for biosynthetic reactions (9, 30). Mitochondria also control decisions for life and death. Changes in mitochondrial membrane permeability lead to the release of proapoptotic mediators that can kill cells with DNA damage or activated oncogenes (16). In this way, mitochondria control one of the major tumor suppressor responses: apoptosis (27). Some oncogenes, such as RasV12, STAT5, and Bcl2, have antiapoptotic activity, and some cell types have a high apoptosis threshold. Another tumor suppressor response, called cellular senescence, serves as a fail-safe mechanism against the transforming activity of antiapoptotic oncogenes (29, 40, 43). However, currently it is unknown whether mitochondria also can play a role in oncogene-induced senescence (OIS).OIS is phenotypically similar to the senescence response triggered by short telomeres, also known as replicative senescence (6). Replicative senescence is, in essence, the consequence of a DNA damage response triggered by short telomeres (11). OIS also involves the DNA damage response (2, 15, 28), but the mechanism of DNA damage and the contribution of mitochondria to it are unclear. It has been demonstrated that mitochondria play a critical role in replicative senescence, and several mitochondrial changes, including an increase in the production of reactive oxygen species (ROS), were reported in cells with short telomeres (34, 35). Mitochondrion-derived ROS contribute to the senescent phenotype by damaging the DNA (35) and therefore amplifying the DNA damage signals originally caused by short telomeres. We reasoned that a similar amplifying mechanism involving the mitochondria could operate in cells expressing oncogenes.Here, we use Ha-RasV12, an oncogenic allele of Ha-Ras, to study the role of mitochondria in OIS. RasV12 is a very important human oncogene and was the first linked to the senescence program (43). We report that oncogenic ras induces an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial superoxide production before any sign of senescent cell cycle arrest. With time, these mitochondrial changes evolved into a severe mitochondrial dysfunction characterized by a further increase in ROS production, the accumulation of depolarized mitochondria around the cell nucleus, a decrease in ATP, and the activation of AMPK. The mechanism of the increase in mitochondrial mass and ROS in response to oncogenic ras was found to be dependent on either p53 or Rb. In addition, direct interference with mitochondrial functions by downregulating the mitochondrial Rieske iron sulfur protein (RISP) or by using pharmacological inhibitors of oxidative phosphorylation induced senescence. We suggest that the senescence effector mechanism acting downstream of p53 and Rb involves mitochondrial dysfunction.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-β induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-β promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.In the battle of hosts and microbes, the innate immune system uses pathogen recognition receptors (PRRs) to sense pathogen-associated molecular patterns (23). There are several functionally distinct classes of PRRs, such as the transmembrane (TM) Toll-like receptors (TLRs) and the intracellular retinoic acid-inducible gene I (RIG-I)-like helicase (RLH) receptors (15, 23, 25, 38). RLHs, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), comprise an N-terminal caspase recruitment domain (CARD), a middle DEXD/H box RNA helicase domain, and a C-terminal domain. RLHs sense intracellular viral RNA and initiate an antiviral interferon (IFN) response (1, 43). RIG-I binding to viral RNA triggers conformational changes that expose the CARD for subsequent signaling (42). The adaptor molecule providing a link between RIG-I and downstream events was identified independently by four research groups as a mitochondrial CARD-containing protein, which was named mitochondrial antiviral signaling protein (MAVS) (34), IFN-β promoter stimulator 1 (IPS-1) (12), virus-induced signaling adaptor (VISA) (40), and CARD adaptor-inducing IFN-β (Cardif) (24). We refer to this adaptor as MAVS in this paper. MAVS transduces signals from RIG-I through CARD-CARD interactions, which then lead to interferon regulatory factor 3 (IRF-3) and NF-κB activation of IFN-β induction through a signaling cascade involving IKKα/β/γ, IKKɛ, and TBK1 (15). Recently, a protein termed STING (11) or MITA (47) was identified as a mediator that acts downstream of RIG-I and MAVS and upstream of TBK1.MAVS protein contains an N-terminal CARD required for signaling, a proline-rich domain that interacts with TRAF3, and a C-terminal TM region that targets MAVS to the mitochondrial outer membrane (29). Several cellular and viral proteins target MAVS in the attenuation of the IFN induction pathway. Cleavage of MAVS by hepatitis C virus (HCV) and hepatitis A virus (HAV) proteases, at residues C508 (18, 24) and Q428 (41), respectively, results in the loss of MAVS mitochondrial localization, thereby disrupting its function in IFN induction. Another mitochondrial outer membrane protein, NLRX1, can sequester MAVS from its association with RIG-I and act as a negative regulator of the IFN pathway (28). MAVS was recently found to be cleaved and inactivated by caspases during apoptosis (31, 33).The caspases are a well-known family of cysteinyl aspartate-specific proteases. The diverse roles of caspases in the cell cycle, proliferation, differentiation, cytokine production, innate immune regulation, and microbial infection suggest various functions of caspases beyond apoptosis (13, 14). The caspases can be separated into two subfamilies, namely, the cell death and inflammation subfamilies. In response to apoptotic stimuli, the initiators caspase-2, -8, -9, and -10 and effectors caspase-3, -6, and -7 mediate cell death events. Caspase-1, -4, -5, and -12 are known as the inflammatory caspases. Caspase-1 is involved in the cleavage and maturation of cytokines (8, 17). Caspase-8 and -10 were discovered as essential components that mediate antiviral signaling (37). Caspase-1 and -3 are activated in innate immune signaling (32). These findings indicate that caspases are involved in the regulation of innate immunity, in addition to their well-known apoptotic role. However, the details of how caspases are activated, the role of caspase activation, and how caspases manipulate the signaling pathways in innate immunity are still obscure.The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Infections with flaviviruses, such as dengue virus (DEN), Japanese encephalitis virus, and West Nile virus, are emerging worldwide. DEN triggers IFN-β through a molecular mechanism involving the RIG-I/MAVS signaling pathway (5, 20). In this study, we found that MAVS is cleaved during DEN serotype 2 (DEN-2) infection, in a caspase-dependent manner; this contrasts with viral protease-dependent cleavage of MAVS during infection with HCV and HAV. In a cell-free caspase assay system, MAVS was cleaved at residue D429 by caspase-1 and caspase-3. Cleaved MAVS failed to induce IFN production and caspase activation, and overexpression of MAVS also triggered caspase activation, which then negatively regulated its own function. Importantly, the role of MAVS in viral infection was verified by knockdown of MAVS expression. We discuss the possible regulatory mechanisms of MAVS and the biological significance of this cleavage event by caspases in the context of understanding how these apoptosis-related proteases might achieve cross talk with the innate immune pathway during viral infection.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.