Eng Shang Huang

Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immune-compromised individuals, including organ transplant recipients and individuals with AIDS. It is also a leading cause of virus-associated birth defects and is associated with atherosclerosis and coronary restenosis. HCMV initiates infection and intracellular signalling by binding to its cognate cellular receptors and by activating several signalling pathways including those mediated by mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, interferons, and G proteins. But a cellular receptor responsible for viral entry and HCMV-induced signalling has yet to be identified. Here we show that HCMV infects cells by interacting with epidermal growth factor receptor (EGFR) and inducing signalling. Transfecting EGFR-negative cells with an EGFR complementary DNA renders non-susceptible cells susceptible to HCMV. Ligand displacement and crosslinking analyses show that HCMV interacts with EGFR through gB, its principal envelope glycoprotein. gB preferentially binds EGFR and EGFR–ErbB3 oligomeric molecules in Chinese hamster ovary cells transfected with erbB family cDNAs. Taken together, these data indicate that EGFR is a necessary component for HCMV-triggered signalling and viral entry.

Integrin αvβ3 is a coreceptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin αvβ3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and αvβ3, respectively, to initiate viral entry and signaling. αvβ3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and αvβ3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and αvβ3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.

E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection.

The adenovirus immediate-early protein E1A activates the adenovirus E2 promoter and several cellular gene promoters through transcription factor E2F. The immediate-early proteins of human cytomegalovirus (HCMV) can complement an E1A-deficient adenovirus mutant and activate the adenovirus E2 promoter. HCMV also has been shown to activate the adenovirus E2 promoter. On the basis of these findings, we have investigated whether HCMV can activate the promoter of the cellular dihydrofolate reductase (DHFR) gene, which requires E2F binding for maximal promoter activity. We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site. We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an infection-specific multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells. Several antibodies against HCMV immediate-early gene products had no effect on this infection-specific complex. Subsequently, the complex was found to contain E2F, cyclin A, p33cdk2, and p107 and to be similar to S-phase-specific complexes that recently have been identified in several cell types. A functional role for the binding of the cyclin A-p33cdk2 complex to cellular gene promoters has yet to be demonstrated; however, HCMV infection causes the induction of both cellular DNA replication and transcription of growth-related genes containing E2F sites in their promoters. The findings described above therefore may relate to both of these effects of HCMV infection. We also provide evidence that some of the molecular events associated with adenovirus infection are different from those associated with HCMV infection.

Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3).

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.

CYTOMEGALOVIRUS D.N.A. AND ADENOCARCINOMA OF THE COLON: EVIDENCE FOR LATENT VIRAL INFECTION

The cause of adenocarcinoma of the colon has not been proven and a viral association has not been reported with this disease. A sensitive biochemical technique for the detection of viral nucleic acid was used to determine whether one type of herpesvirus, namely a human strain of cytomegalovirus, was uniquely present in tumour tissue. 24 specimens of colon, obtained at surgery from 14 patients, were analysed by membrane CR.N.A.-D.N.A. hybridisation. 4 of 7 tumours of the colon were definitely positive for C.M.V. D.N.A. (2greater than or equal to 2 genome-equivalents/cell), or repeatedly showed more than 1 genome-equivalent/cell. Macroscopically normal colon 5 cm from the tumour, as well as histologically normal and abnormal colon from a control population with Crohns disease were uniformly negative for C.M.V. D.N.A., except for 1 specimen which was macroscopically normal tissue from a patient with carcinoma of the colon. Among patients with conditions that predispose to adenocarcinoma of the colon, 1 of 2 patients with familial polyposis and 1 of 3 with ulcerative colitis harboured in their colon greater than or equal to 2 genome-equivalents of C.M.V. D.N.A. per cell, and another ulcerative colitis patient had 1-2 genome-equivalents/cell.

Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences.

Our previous work demonstrated that following human cytomegalovirus (HCMV) infection of fibroblasts, there was a protein-protein interaction between the HCMV IE1-72 immediate-early (IE) protein and the cellular p107 protein which resulted in the alleviation of p107-mediated transcriptional repression of E2F-responsive promoters. In a further characterization of this interaction, we now show that IE1-72 binds to the N-terminal portion of p107, not the C-terminal pocket region that binds E2F-4, and where a number of other viral gene products bind. Additionally, we show that exons 2 and 3 of IE1-72 are required for binding to p107. After mapping the binding domains, we next wanted to address the additional functional consequences of this interaction. It is well known that p107 can negatively regulate cell growth. To examine whether IE1-72 can also overcome this growth suppression, we transfected and infected or cotransfected various constructs into SAOS-2 cells. We showed that infection of SAOS-2 cells was capable of alleviating p107-mediated growth suppression. Additionally, we showed that IE1-72 alone is capable of overcoming p107-mediated growth arrest. Alleviation of this repression by IE1-72 is dependent on the protein-protein interaction between p107 and IE1-72 as deletion mutants of either protein which lack the identified binding domains fail to achieve this effect. These data indicate that the IE1-72 protein is capable of overcoming p107-mediated blocks in cellular proliferation, events that occur in both productive and non-productive HCMV infections.

Interactions between Human Cytomegalovirus IE1-72 and Cellular p107: Functional Domains and Mechanisms of Up-Regulation of Cyclin E/cdk2 Kinase Activity

ABSTRACT Previous work has demonstrated that the human cytomegalovirus IE1-72 protein is able to bind to the N terminus of p107, and IE1-72 alone is sufficient for alleviation of p107-mediated cell growth suppression. However, the mechanism of this alleviation is unclear. Here, we show that IE1-72 can alleviate p107 inhibition of cyclin E/cdk2 kinase activity. We cotransfected various IE1-72 and p107 constructs into C33A cells and demonstrated that IE1-72 could activate the kinase activity of cyclin E/cdk2. Conversely, IE2-86 did not activate this activity, suggesting that the interaction between p107 and IE1-72 and the subsequent kinase activation are specific. By the use of a series of deletion and point mutants of IE1-72 and p107, we observed that a mutation of the loop region of helix-loop-helix-turn-helix in exon 3 of IE1-72 as well as a mutation of the leucine zipper-2 region in exon 4 of IE1-72 abolished binding to p107. In addition, these two IE1-72 mutants did not alleviate p107 inhibition of cyclin E/cdk2 kinase activity and also failed to alleviate p107 inhibition of the E2F-responsive promoter. Meanwhile, deletion of the N-terminal aa 1 to 175 of p107 abolished both p107 binding with IE1-72 and p107 inhibition of cyclin E/cdk2 kinase activity. This result confirms that the N-terminus aa 1 to 175 region of p107 is a common region where both IE1-72 protein and cyclin E/cdk2 bind. We propose a mechanism in which binding of IE1-72 to p107 displaces cyclin E/cdk2 from p107. Once released from p107, cyclin E/cdk2 is able to function as an active kinase.

Disparate effects of two herpesviruses immediate-early gene trans-activators on the HIV-1 LTR

Abstract The BMLF1 region of the Epstein-Barr virus (EBV) genome and the immediate-early (IE) region of human cytomegalovirus (HCMV) both encode proteins which can trans -activate heterologous promoter/chloramphenicol acetyl transferase (CAT) constructs, including a human immunodeficiency virus type-1 promoter/CAT construct. We demonstrate here that this trans -activation by the EBV BMLF1 gene product, which we have previously shown to be largely post-transcriptional, is reporter gene dependent. In contrast, trans -activation by the HCMV-IE gene product(s), previously shown to be mediated at the RNA level, is seen regardless of whether CAT, human growth hormone, or β-galactosidase is used as the reporter gene. Mutational analysis revealed no specific cis -acting sequences within the HIV-1 promoter which were required for trans -activation by the HCMV-IE gene product(s).