Requirements for the association of adenovirus type 2 E3/19K wild-type and mutant proteins with HLA antigens

The E3/19K protein of human adenovirus type 2 is a resident of the endoplasmic reticulum (ER). Immediately after synthesis, it associates with major histocompatibility complex class I antigens and prevents their intracellular transport and cell surface expression. We have generated several C-terminal deletion mutants of the E3/19K protein that are preterminated at various positions on both sides of the membrane-spanning segment of the protein. One of these mutants is terminated at the luminal side of the membrane (M310), and two are terminated in the hydrophobic segment (M374 and M392), whereas mutant M621 is terminated on the cytoplasmic side of the ER membrane. The M310, M374, and M392 mutants are soluble proteins. They do not associate with HLA antigens in transfected 293 cells, and they are, to some extent, secreted into the medium. The M621 mutant protein is integrated in the ER membrane, associates immediately after its synthesis with HLA antigens, and exits from the ER. By using either an in vitro translation system supplemented with microsomes or overexpression in insect cells, we showed that M374 and E3/19K are able to associate with HLA antigens. These results indicate that the conformation of the luminal part of the E3/19K protein is not grossly altered by the mutations. Rapid transport of the M374 mutant out of the ER and partial degradation of this protein may prevent the interaction with HLA class I antigens in transfected 293 cells.     

The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K protein binds to peptide-filled and peptide-deficient HLA-A*1101 molecules

E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 +/- 10 nM for peptide-filled molecules and of about 10 microM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.     

Allele- and locus-specific recognition of class I MHC molecules by the immunomodulatory E3-19K protein from adenovirus

The E3-19K protein from human adenoviruses (Ads) retains class I MHC molecules in the endoplasmic reticulum. As a consequence, the cell surface expression of class I molecules is suppressed, allowing Ads to evade immune surveillance. Using native gel electrophoresis, gel filtration chromatography, and surface plasmon resonance, we show that a soluble form of the Ad type 2 (Ad2) E3-19K protein associates with HLA-A and -B molecules; equilibrium dissociation constants were in the nanomolar range and approximately 2.5-fold higher affinity for HLA-A (-A*0201, -A*0301, -A*1101, -A*3301, and -Aw*6801) relative to HLA-B (-B*0702 and -B*0801) molecules. Among the alleles of the HLA-A locus examined, HLA-A*3101 associated approximately 15-fold less avidly with soluble E3-19K. Soluble E3-19K interacted only very weakly with HLA-Cw*0304, and no interaction with HLA-Cw*0401 could be detected under identical conditions. Site-directed mutagenesis and flow cytometry demonstrated that MHC residue 56 plays a critical role in the association and endoplasmic reticulum retention of HLA-A molecules by E3-19K. This delineates the spatial environment around residue 56 as a putative E3-19K interaction surface on class I molecules. Overall, our data imply that a link may exist between host genetic factors and the susceptibility of individuals to Ad infections.     

The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus

The association between the retinoblastoma protein (p105-RB) and either the large T antigen of SV40 or the E1A proteins of adenovirus is thought to be an important step in transformation by these viral oncogenes. E1A and large T antigen share a small region of amino acid homology that is necessary for high affinity binding with p105-RB. Mutations of this homology region were shown to reduce drastically the frequency of transformation mediated by the E1A or large T oncogenes. Previously, this small region in E1A was shown to be sufficient for interaction with a second cellular protein of 107,000 daltons (107K). Here we show that in human cells, the large T antigens of SV40 or JC virus also form complexes with 107K. Demonstration of complexes between 107K and the large T antigens of SV40 and JC virus suggests that these associations may represent another component of a common mechanism for transformation between adenoviruses and polyoma viruses.     

The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.     

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.     

A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.     

An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens

The adenovirus type 2 encoded protein E3/19K binds to human histocompatibility class I antigens (HLA). This association occurs both in adenovirus-infected cells and in cells that have been transfected with the gene encoding the E3/19K protein. The formation of the HLA-E3/19K complex prevents the HLA antigens from being correctly processed by inhibiting their terminal glycosylation. This effect is specific for HLA antigens and does not generally involve the glycosyltransferases. Furthermore, the HLA-E3/19K association dramatically reduces the cell surface expression of the HLA antigens. This reduced level of antigens might influence the cytotoxic T cell response. Therefore, our results show a possible molecular mechanism whereby adenoviruses, and perhaps other viruses, delay or escape the cellular immune system of the host.     

Interaction of the adenovirus type 5 E4 Orf3 protein with promyelocytic leukemia protein isoform II is required for ND10 disruption

Nuclear domain 10 (ND10s), or promyelocytic leukemia protein (PML) nuclear bodies, are spherical nuclear structures that require PML proteins for their formation. Many viruses target these structures during infection. The E4 Orf3 protein of adenovirus 5 (Ad5) rearranges ND10s, causing PML to colocalize with Orf3 in nuclear tracks or fibers. There are six different PML isoforms (I to VI) present at ND10s, all sharing a common N terminus but with structural differences at their C termini. In this study, PML II was the only one of these six isoforms that was found to interact directly and specifically with Ad5 E4 Orf3 in vitro and in vivo; these results define a new Orf3 activity. Three of a series of 18 mutant Orf3 proteins were unable to interact with PML II; these were also unable to cause ND10 rearrangement. Moreover, in PML-null cells that contained neoformed ND10s comprising a single PML isoform, only ND10s formed of PML II were rearranged by Orf3. These data show that the interaction between Orf3 and PML II is necessary for ND10 rearrangement to occur. Finally, Orf3 was shown to self-associate in vitro. This activity was absent in mutant Orf3 proteins that were unable to form tracks and to bind PML II. Thus, Orf3 oligomerization may mediate the formation of nuclear tracks in vivo and may also be important for PML II binding.     

The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus-2 is microtubule binding.

The signal for retention in the endoplasmic reticulum of the E3/19K protein of adenovirus type 2 is located within the carboxyl-terminal cytoplasmic extension. A synthetic peptide corresponding to this sequence showed affinity for beta-tubulin, could promote tubulin polymerization in vitro, and bound to taxol-polymerized microtubules. When compared with the microtubule binding sequences from two microtubule-associated proteins (MAPs; MAP2 and tau), we found similarities suggesting that the cytoplasmic tail might bind to tubulin/microtubules in a MAPs-like fashion. A synthetic peptide corresponding to the cytoplasmic tail of an E3/19K deletion mutant not retained in the endoplasmic reticulum was also tested. It had the same net charge but did not promote tubulin polymerization in vitro nor did it show measurable affinity for tubulin or microtubules. This indicates that binding to microtubules is important for retention of the E3/19K protein in the endoplasmic reticulum.     

Reovirus type 3 binds to antagonist domains of the beta-adrenergic receptor.

Reovirus type 3 interfered with the binding of beta-adrenergic antagonist ligands to receptors on Y1 adrenal, C6 glioma, and mouse L cells. This inhibition of beta-adrenergic binding was dose related. Reovirus did not interfere with dopaminergic binding or isoproterenol-induced activation of adenylate cyclase. In addition, reovirus infected Y1 cells, which bind beta-adrenergic antagonist ligands but lack agonist-induced activity. These results suggest that reovirus infection is initiated by binding to antagonist (nonfunctional) domains of the adrenergic receptor complex.     

Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3)

Huntington''s disease (HD) is a neurodegenerative disorder characterized by progressive neuronal death in the basal ganglia and cortex. Although increasing evidence supports a pivotal role of mitochondrial dysfunction in the death of patients'' neurons, the molecular bases for mitochondrial impairment have not been elucidated. We provide the first evidence of an abnormal activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNip3) in cells expressing mutant Huntingtin. In this study, we show an abnormal accumulation and dimerization of BNip3 in the mitochondria extracted from human HD muscle cells, HD model cell cultures and brain tissues from HD model mice. Importantly, we have shown that blocking BNip3 expression and dimerization restores normal mitochondrial potential in human HD muscle cells. Our data shed light on the molecular mechanisms underlying mitochondrial dysfunction in HD and point to BNip3 as a new potential target for neuroprotective therapy in HD.     

The 55K protein on the 5'' termini of adenovirus type 2 DNA is unrelated to virus-coded candidate transformation proteins (E1-53K, E1-40K-50K) and DNA-binding proteins (E2-42K/47K/73K).

A polypeptide of 55,000 daltons (55K) is linked, probably covalently, to the K' termini of adenovirus type 2 DNA. The 55K polypeptide is synthesized during early stages of infection (T. Yamashita, M. Arens, and M. Green, J. Virol. 30: 497-507, 1979) and thus may function in viral DNA replication, gene regulation, or cell transformation. Several virus-coded early polypeptides have been identified that could correspond to the terminal 55K, including the E1-40K-50K and E1-53K candidate transformation polypeptides and the E2-42K/47K/73K single-stranded DNA-binding polypeptide. We show here that two-dimensional tryptic [35S]methionine-peptide maps of the terminal 55K differ completely from [35S]methionine-peptide maps of four related E1-40K-50K polypeptides, the E1-53K, and the related E2-42K, E2-47K, and E2-73K polypeptides. We conclude that the terminal 55K polypeptide does not correspond to any of the known virus-coded early polypeptides.