Byung Yoon Ahn

Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen

Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N‐terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N‐terminal region (residues 7–117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four‐helix bundle, and residues from helices α2 and α4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ‐1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb.

Ability of the Human Cytomegalovirus IE1 Protein To Modulate Sumoylation of PML Correlates with Its Functional Activities in Transcriptional Regulation and Infectivity in Cultured Fibroblast Cells

ABSTRACT In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(Δ290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.

Novel mucosal immunization with polysaccharide–protein conjugates entrapped in alginate microspheres

A novel mucosal immunization was examined using biocompatible and biodegradable alginate microspheres containing a conjugate of polysaccharide antigen and cholera toxin B subunit (CTB). In order to prepare the alginate microspheres with diameters of less than 5 microm, a new diffusion-controlled interfacial gelation technique was developed. Also, in order to improve the mucosal immune response, a pneumococcal capsular polysaccharide type 19 (PS19) was conjugated to the CTB (PS19-CTB). This conjugate was subsequently encapsulated into the alginate microspheres. The loading content of PS19-CTB to the alginate microspheres was 60%. An in vitro sustained release pattern was observed with the antigen-loaded microspheres, showing 80% antigen release within one day. Mucosal and systemic immunities following oral immunization with the alginate microspheres were studied. Balb/c mice were immunized perorally three times at intervals of two weeks. Peroral immunization with 25 microg of PS19-CTB entrapped in the alginate microspheres evoked both the mucosal IgA and systemic IgM responses to PS19 in small intestine and in sera, respectively. The results suggest that both the mucosal and systemic antibody responses could be induced by oral administration of the PS19-CTB antigen entrapped in alginate microspheres.

Cross-Protective Immunity of Mice Induced by Oral Immunization with Pneumococcal Surface Adhesin A Encapsulated in Microspheres

ABSTRACT The global use of a capsular polysaccharide-based pneumococcal vaccine has been limited because of serotype-specific protection and poor effectiveness in individuals with low immunocompetency. The mucosal immune system develops earlier in infants and lasts longer in the elderly than does the systemic immune system. Furthermore, mucosal immunization is beneficial for AIDS patients, because human immunodeficiency virus-infected subjects can develop normal mucosal antibody responses even in late clinical phases. For these reasons, we evaluated recombinant pneumococcal surface adhesin A (rPsaA) of Streptococcus pneumoniae in terms of cross-protective immune responses after oral delivery. Encapsulated rPsaA provided higher immunogenicity than naked rPsaA. Coencapsulation or codelivery of the cholera toxin (CT) B subunit (CTB) and CT also increased the immunogenicity of rPsaA. Cross-protective immunities against five strains of S. pneumoniae (types 4, 6B, 14, 19F, and 23F) were induced after oral immunization with microencapsulated rPsaA. Lung colonization and septicemia caused by the five serotypes were significantly inhibited by oral immunization with microencapsulated rPsaA. These results suggest that rPsaA coencapsulated with CTB can be used as an oral vaccine to induce cross-protective immunity for the prevention of pneumococcal infection.

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection.

Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by alpha v beta3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infection and pathogenesis.

Structural and Functional Dissection of Human Cytomegalovirus US3 in Binding Major Histocompatibility Complex Class I Molecules

ABSTRACT The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.

Cell-surface Receptor for Complement Component C1q (gC1qR) Is a Key Regulator for Lamellipodia Formation and Cancer Metastasis

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

Proteome analysis of adipocyte lipid rafts reveals that gC1qR plays essential roles in adipogenesis and insulin signal transduction.

Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent‐resistant lipid raft proteins from 3T3‐L1 preadipocytes and adipocytes by 2‐DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte‐specific proteins was globular C1q receptor (gC1qR), an acidic 32 kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin‐induced activation of insulin receptor, insulin receptor substrate‐1 (IRS‐1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling.

PKR protein kinase is activated by hepatitis C virus and inhibits viral replication through translational control.

Hepatitis C virus (HCV) infection is currently treated with IFNalpha-based therapy but little is known how IFNalpha inhibits HCV replication. We show here that HCV JFH1 infection of human hepatoma Huh-7 cells leads to the activation of IFN-inducible protein kinase PKR and phosphorylation of the translation initiation factor eIF2alpha. Compared to a control cell HCV replication was significantly elevated in a PKR-knockdown cell, giving rise to a 10-fold higher viral titer, and was less sensitive to IFNalpha treatment. Conversely, transient expression of PKR inhibited HCV replication in a kinase-dependent manner with concomitant increase of eIF2alpha phosphorylation. Further, expression of a phospho-mimetic eIF2alpha mutant moderately inhibited HCV replication. Together, these results demonstrate that PKR is activated by HCV infection and plays a critical antiviral role through inhibition of viral protein translation.

Surveillance study (2000 to 2001) of G- and P-type human rotaviruses circulating in South Korea.

ABSTRACT Human rotavirus VP4 and VP7 gene sequences were amplified by reverse transcription-PCR from 53% (322 of 607) of fecal specimens collected from children with severe diarrhea who visited hospitals in six urban areas of South Korea in 2000 and 2001. G2 was the most frequently found G type (constituted 50.6%), followed by G1 (30.1%) and G4 (13.0%). Although the P types of high incidence were P[4] (53.1%) and P[8] (21.4%), a significant incidence of P[6] (20.2%) was also noticeable. The commonest G- and P-type combination found in this study was G2P[4], rather than G1P[8], the most prevalent type known worldwide.