FAdV-4-induced ferroptosis affects fat metabolism in LMH cells.

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

Identification of Adenovirus E1B-55K Interaction Partners through a Common Binding Motif.

The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

The Cajal body protein p80-coilin forms a complex with the adenovirus L4-22K protein and facilitates the nuclear export of adenovirus mRNA.

IMPORTANCE: The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus.

Adenovirus infection controls processing bodies to stabilize AU-rich element-containing mRNA.

In the adenovirus-infected cells, virus mRNAs are selectively exported to the cytoplasm by virus early gene products to facilitate virus replication. We previously showed AU-rich elements (AREs) containing mRNAs are exported to the cytoplasm and stabilized in infected cells. Here, we analyzed ribonucleoprotein (RNP) granules in the cytoplasm that are involved in mRNA degradation to elucidate the mechanism of ARE-mRNA stabilization in adenovirus infected cells. Our findings showed that processing bodies (PBs) aggregate, then almost all PBs are translocated to aggresomes formed by adenoviral gene products during the late phase of infection. Furthermore, E4orf3 was required for the PBs translocation, and the same domains of E4orf3-mutants required to change the form of promyelocytic leukemia bodies were also needed for PBs translocation. Luciferase activity showed that these domains were critical for miRNA- and ARE-mediated mRNA decay. These findings suggest that adenovirus changes the behavior of PBs to prevent ARE-mRNA downregulation.

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape.

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.

DNA-PK phosphorylation at Ser2056 during adenovirus E4 mutant infection is promoted by viral DNA replication and independent of the MRN complex.

Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.

CRM1 Promotes Capsid Disassembly and Nuclear Envelope Translocation of Adenovirus Independently of Its Export Function.

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.

An Adenovirus early region 4 deletion mutant induces G2/M arrest via ATM activation and reduces expression of the mitotic marker phosphorylated (ser10) histone 3.

Adenovirus (Ad) type 5 (Ad5) early region 4 (E4) proteins inhibit the DNA damage response (DDR) including activation of the DDR kinase ATM and its substrates, which can induce G2/M cell cycle arrest. Infection with Ad5 or the E4 deletion mutant H5dl1007 (1007) resulted in the accumulation of post G1 cells with > 2 N cellular DNA content. A greater fraction of cells with 4 N DNA content was observed in 1007 infections compared to Ad5; this population was dependent on activation of ATM. G2/M checkpoint kinases, phosphorylated Chk2 (pChk2), and phosphorylated Cdk1 (pCdk1) were upregulated in 1007 infections, and 1007 showed reduced levels of the mitosis marker phosphorylated (Ser10) histone 3 compared to Ad5. Our results show that E4 mutant activation of ATM induces G2/M arrest via activation of checkpoint kinases, thereby contributing to viral-mediated regulation of the cell cycle.

The RGD-binding integrins αvß6 and αvß8 are receptors for mouse adenovirus-1 and -3 infection.

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvß6 and αvß8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mß6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvß6-positive CMT-93 cells, whereas mß8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvß8-positive M000216 cells. Soluble integrin αvß6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvß6/αvß8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvß6/ß8, where the distal leucine residue dips into a hydrophobic pocket of ß6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvß6/ß8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvß6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes.

The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.

Fowl Adenovirus Serotype 4 Induces Hepatic Steatosis via Activation of Liver X Receptor-α.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic virus that causes severe hepatic damage characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal necrosis in hepatocytes. Many aspects of FAdV-4 infection and pathogenesis, however, remain unknown. Here, we found that FAdV-4-induced hepatic injury is accompanied by the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in FAdV-4-infected chickens. Significant upregulation of adipose synthesis-related genes, such as liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding protein-1c (SREBP-1c), and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation- and lipid decomposition-related genes were observed in the infected chickens. FAdV-4 infection in cultured leghorn male hepatoma (LMH) cells caused similar signs of steatosis, with alterations in various lipogenesis-related genes. We eliminated the effect of LXR-α activation on FAdV-4-induced steatosis and found that treatment with an LXR-α antagonist (SR9243) and RNA interference (small interfering RNA targeting LXR-α [Si-LXR-α]) decreased the number of oil droplets and the accumulation of lipogenic genes, but treatment with an LXR-α agonist (T0901317) increased the number of oil droplets and the accumulation of lipogenic genes in the cells. Additionally, SR9243 treatment or Si-LXR-α transfection led to significant reductions in viral DNA level, protein expression, and virus production, whereas T0901317 treatment caused significant increases in viral DNA level, protein expression, and virus production. However, inhibition of SREBP-1c activity had no significant effect on virus production. Collectively, these results indicated that FAdV-4-induced steatosis involves activation of the LXR-α signaling pathway, which might be a molecular mechanism underlying the hepatic injury associated with FAdV-4 infection.IMPORTANCE Fowl adenovirus serotype 4 (FAdV-4) is an important hepatotropic adenovirus in chicken, but the underlying mechanism of FAdV-4-induced hepatic injury remains unclear. We report here that infection with FAdV-4 induced the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in the livers of chickens. FAdV-4-induced steatosis might be caused by a disrupted balance of fat metabolism, as evidenced by differential regulation of various lipase genes. The significant upregulation of liver X receptor-α (LXR-α) prompted us to investigate the interplay between LXR-α activation and FAdV-4-induced steatosis. Treatment with an agonist, an antagonist, or RNA interference targeting LXR-α in cultured leghorn male hepatoma (LMH) cells indicated that FAdV-4-induced steatosis was dependent upon LXR-α activation, which contributed to virus replication. These results provide important mechanistic insights, revealing that FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway and highlighting the therapeutic potential of strategies targeting the LXR-α pathway for the treatment of FAdV-4 infection.

Adenoviral E1A Exploits Flexibility and Disorder to Target Cellular Proteins.

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.

En Guard! The Interactions between Adenoviruses and the DNA Damage Response.

Virus-host cell interactions include several skirmishes between the virus and its host, and the DNA damage response (DDR) network is one of their important battlegrounds. Although some aspects of the DDR are exploited by adenovirus (Ad) to improve virus replication, especially at the early phase of infection, a large body of evidence demonstrates that Ad devotes many of its proteins, including E1B-55K, E4orf3, E4orf4, E4orf6, and core protein VII, and utilizes varied mechanisms to inhibit the DDR. These findings indicate that the DDR would strongly restrict Ad replication if allowed to function efficiently. Various Ad serotypes inactivate DNA damage sensors, including the Mre11-Rad50-Nbs1 (MRN) complex, DNA-dependent protein kinase (DNA-PK), and Poly (ADP-ribose) polymerase 1 (PARP-1). As a result, these viruses inhibit signaling via DDR transducers, such as the ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, to downstream effectors. The different Ad serotypes utilize both shared and distinct mechanisms to inhibit various branches of the DDR. The aim of this review is to understand the interactions between Ad proteins and the DDR and to appreciate how these interactions contribute to viral replication.

Adenovirus-mediated ubiquitination alters protein-RNA binding and aids viral RNA processing.

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

Adenovirus targets transcriptional and posttranslational mechanisms to limit gap junction function.

Adenoviruses are responsible for a spectrum of pathogenesis including viral myocarditis. The gap junction protein connexin43 (Cx43, gene name GJA1) facilitates rapid propagation of action potentials necessary for each heartbeat. Gap junctions also propagate innate and adaptive antiviral immune responses, but how viruses may target these structures is not understood. Given this immunological role of Cx43, we hypothesized that gap junctions would be targeted during adenovirus type 5 (Ad5) infection. We find reduced Cx43 protein levels due to decreased GJA1 mRNA transcripts dependent upon ß-catenin transcriptional activity during Ad5 infection, with early viral protein E4orf1 sufficient to induce ß-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation events consistent with altered gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation and complementary studies in human induced pluripotent stem cell derived-cardiomyocytes reveal Cx43 gap junction remodeling by reduced ZO-1 complexing. These findings reveal specific targeting of gap junction function by Ad5 leading to loss of intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts.

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV.

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Turkey adenovirus 3, a siadenovirus, uses sialic acid on N-linked glycoproteins as a cellular receptor.

Turkey adenovirus 3 (TAdV-3) is the causative agent of an immune-mediated disease in turkeys, haemorrhagic enteritis, through targeting B lymphocytes. In the present study, we investigated the role of sialic acid in TAdV-3 entry and characterized the structural components of TAdV-3 receptor(s) on RP19, B lymphoblastoid cells. Removal of the cell-surface sialic acids by neuraminidases or blocking of sialic acids by wheat germ agglutinin lectin reduced virus infection. Pre-incubation of cells with Maackia amurensis lectin or Sambucus nigra agglutinin resulted in virus reduction, suggesting that TAdV-3 uses both α2,3-linked and α2,6-linked sialic acids as attachment receptor. Virus infectivity data from RP19 cells treated with sodium periodate, proteases (trypsin or bromelain) or metabolic inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, tunicamycin, or benzyl N-acetyl-α-d-galactosaminide) indicated that N-linked, but not O-linked, carbohydrates are part of the sialylated receptor and they are likely based on a membrane glycoprotein, rather than a glycolipid. Furthermore, our data, in conjunction with previous findings, implies that the secondary receptor for TAdV-3 is a protein molecule since the inhibition of glycolipid biosynthesis did not affect the virus infection, which was rather reduced by protease treatment. We can conclude that terminal sialic acids attached to N-linked membrane glycoproteins on B cells are used for virus attachment and are essential for successful virus infection.

The coxsackievirus and adenovirus receptor: virological and biological beauty.

The coxsackievirus and adenovirus receptor (CAR) is an essential multifunctional cellular protein that is only beginning to be understood. CAR serves as a receptor for many adenoviruses, human group B coxsackieviruses, swine vesicular disease virus, and possibly other viruses. While named for its function as a viral receptor, CAR is also involved in cell adhesion, immune cell activation, synaptic transmission, and signaling. Knockout mouse models were first to identify some of these biological functions; however, tissue-specific model systems have shed light on the complexity of different CAR isoforms and their specific activities. Many of these functions are mediated by the large number of interacting proteins described so far, and several new putative interactions have recently been discovered. As antiviral and gene therapy strategies that target CAR continue to emerge, future work poised to understand the biological implications of manipulating CAR in vivo is critical.