Design of protein-protein interaction specificity using computational methods and experimental library screening

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Computational design of protein-protein interaction specificity is a powerful tool to examine and expand our understanding about how protein sequence determines interaction specificity. It also has many applications in basic bioscience and biotechnology. One of the major challenges for design is that current scoring functions relying on general physical principles do not always make reliable predictions about interaction specificity. In this thesis I described application of two approaches to address this problem. The first approach sought to improve scoring functions with experimental interaction specificity data related to the protein family of design interest. I used this approach to design inhibitor peptides against the viral bZIP protein BZLF 1. Specificity against design self-interaction was considered in the study. The second approach exploited the power of experimental library screening to characterize a large number of designed sequences at once, increasing the overall probability of identifying successful designs. I presented a novel framework for such library design approach and applied it to the design of anti-apoptotic Bcl-2 proteins with novel interaction specificity toward BH3 peptides. Finally I proposed how these two approaches can be combined together to further enhance our design capabilities.by Tsan-Chou Scott Chen.Ph.D

Mechanisms of immune evasion in Epstein-Barr virus infection

The human herpesvirus Epstein-Barr virus (EBV) is a large DNA virus that infects over 90% of the adult world population. EBV is the causative agent of infectious mononucleosis and EBV infection is associated with various malignancies. EBV establishes lifelong infections in immunocompetent hosts. To counteract the host’s immune defence, EBV acquired numerous immune evasion mechanisms. During latency of EBV, viral protein synthesis is limited or absent, making the virus-infected cells virtually invisible to the immune system. Evasion mechanisms of EBV active during primary infection as well as in reactivation are necessary for establishment of latent infection and prolonged replication. Studying viral evasion not only helps to understand EBV, but also the human immune system. Viral molecules interfering with antigen presentation by HLA I and HLA II have been identified previously, but so far, it was unclear how EBV interferes with the lipid antigen-presenting molecule CD1d. The work described in this thesis shows EBV’s mechanism to interfere with cell surface expression of CD1d. Further, a novel immune evasion molecule that obstructs antigen-presentation during the late lytic phase of EBV infection was identified and its working mechanism was unravelled. Understanding viral immune evasion mechanisms may aid in developing therapies for EBV-associated diseases

Cloning and characterization of Epstein-Barr virus latent membrane protein 2 (LMP 2) gene.

by Liu Chun Ki, Kevin.Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.Includes bibliographical references (leaves 126-142).Abstracts in English and Chinese.Abstract --- p.iAcknowledgements --- p.iiTable of contents --- p.iiiList of figures --- p.viiiList of tables --- p.xList of abbreviations --- p.xiChapter Chapter 1 --- Introduction Epstein-Barr VirusChapter 1.1 --- History --- p.1Chapter 1.2 --- Classification --- p.2Chapter 1.3 --- Virus and genome structure --- p.3Chapter 1.4 --- Epidemiology --- p.6Chapter 1.4.1 --- Prevalence of infection --- p.6Chapter 1.4.2 --- Modes of transmission --- p.7Chapter 1.5 --- Pathogenesis of EBV --- p.7Chapter 1.5.1 --- "Adsorption, penetration and dissemination" --- p.7Chapter 1.5.2 --- Lytic infection cycle --- p.8Chapter 1.5.3 --- Latent infection cycle --- p.9Chapter 1.5.4 --- Functions of the EBV-specific proteins associated with latent infection cycle proteins --- p.10Chapter 1.5.4.1 --- EBNA1 --- p.10Chapter 1.5.4.2 --- EBNA2 --- p.11Chapter 1.5.4.3 --- "EBNA 3A, 3B and 3C" --- p.11Chapter 1.5.4.4 --- EBNA LP --- p.12Chapter 1.5.4.5 --- LMP1 --- p.13Chapter 1.5.4.6 --- Characteristics of EBV LMP 2 gene --- p.14Chapter 1.5.4.7 --- Functions of LMP 2A --- p.15Chapter 1.5.4.8 --- Functions of LMP 2B --- p.18Chapter 1.6 --- Clinical significance of EBV --- p.20Chapter 1.6.1 --- Infectious mononucleosis (IM) --- p.20Chapter 1.6.2 --- Burkitt's lymphoma (BL) --- p.20Chapter 1.6.3 --- Nasopharyngeal carcinoma (NPC) --- p.21Chapter 1.6.4 --- Hodgkin's lymphoma (HL) --- p.21Chapter 1.7 --- Immune response to EBV infection --- p.22Chapter 1.7.1 --- Humoral immune response --- p.22Chapter 1.7.2 --- Cellular immune response --- p.22Chapter 1.8 --- Diagnosis of EBV infection --- p.26Chapter 1.9 --- Treatment and prevention --- p.27Chapter 1.10 --- Nasopharygneal Carcinoma (NPC) --- p.28Chapter 1.10.1 --- Epidemiology --- p.28Chapter 1.10.2 --- Etiology --- p.28Chapter 1.10.2.1 --- Environmental factor associated with NPC --- p.30Chapter 1.10.2.2 --- Genetic factors associated with NPC --- p.31Chapter 1.10.2.3 --- Association of NPC and EBV --- p.31Chapter 1.10.3 --- Diagnosis ofNPC --- p.32Chapter 1.10.4 --- Treatment --- p.33Chapter 1.11 --- Objective of the project --- p.34Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- EBV-containing cell cultures --- p.35Chapter 2.2 --- Extraction of total RNA --- p.36Chapter 2.2.1 --- Cell lysis --- p.36Chapter 2.2.2 --- Protein digestion --- p.36Chapter 2.2.3 --- DNA digestion --- p.37Chapter 2.2.4 --- Elution of total RNA --- p.37Chapter 2.2.5 --- Purity and electrophoresis analysis of total RNA --- p.38Chapter 2.3 --- First strand cDNA synthesis --- p.38Chapter 2.4 --- PCR amplification of LMP 2 cDNA --- p.39Chapter 2.5 --- Isolation of the PCR amplified LMP 2 cDNA --- p.40Chapter 2.6 --- Purification of the PCR amplified LMP 2 cDNA --- p.41Chapter 2.7 --- Confirmation of the PCR amplified cDNA --- p.42Chapter 2.7.1 --- Nested PCR --- p.42Chapter 2.7.2 --- Restriction enzyme digestion --- p.44Chapter 2.8 --- Ligation of insert LMP 2 cDNA with vector --- p.45Chapter 2.9 --- Transformation of competent cells JM109 --- p.45Chapter 2.10 --- Screening of the recombinant clones --- p.47Chapter 2.11 --- Small scale purification of plasmid DNA --- p.47Chapter 2.12 --- Determination of the size of the insert DNA --- p.48Chapter 2.13 --- DNA sequencing --- p.48Chapter 2.13.1 --- The cycle sequencing reaction --- p.48Chapter 2.13.2 --- Preparation of the acrylamide gel and TBE buffer --- p.51Chapter 2.13.3 --- Running conditions of the electrophoresis --- p.52Chapter 2.13.4 --- "Processing, editing and exporting the sequences" --- p.52Chapter 2.14 --- Data analysis --- p.53Chapter 2.14.1 --- Sequence analysis --- p.53Chapter 2.14.2 --- Amino acid analysis --- p.53Chapter 2.14.3 --- Protein secondary structure analysis --- p.53Chapter 2.14.4 --- Hydrophobicity analysis --- p.54Chapter 2.14.5 --- Isoelectric point analysis --- p.54Chapter Chapter 3 --- ResultsChapter 3.1 --- Cell Cultures --- p.55Chapter 3.2 --- Extraction of total RNA --- p.56Chapter 3.3 --- PCR amplification --- p.61Chapter 3.4 --- Isolation of PCR amplified LMP 2 cDNA --- p.62Chapter 3.5 --- Confirmation of the PCR amplified cDNA --- p.66Chapter 3.5.1 --- Nested PCR --- p.66Chapter 3.5.2 --- Restriction enzyme digestion --- p.71Chapter 3.6 --- Transformation and screening --- p.77Chapter 3.7 --- Extraction of plasmid DNA and its digestion with restriction enzyme --- p.78Chapter 3.8 --- DNA sequencing --- p.83Chapter 3.8.1 --- DNA sequence comparison --- p.84Chapter 3.9 --- Amino acid sequence homology --- p.89Chapter 3.9.1 --- Amino acid sequence comparison --- p.90Chapter 3.10 --- Hydrophobicity analysis --- p.92Chapter 3.10.1 --- Comparison of hydrophobicity of B95-8 derived LMP2 with GeneBank --- p.93Chapter 3.10.2 --- Comparison of hydrophobicity of CB 14022-derived LMP2 with GeneBank --- p.95Chapter 3.10.3 --- Comparison of hydrophobicity of Raji-derived LMP2 with GeneBank --- p.97Chapter 3.11 --- Protein secondary structure analysis --- p.100Chapter 3.11.1 --- Comparison of secondary structure of B95-8-derived LMP2 with GeneBank --- p.100Chapter 3.11.2 --- Comparison of secondary structure of CB 14022-derived LMP2 with GeneBank --- p.100Chapter 3.11.3 --- Comparison of secondary structure of Raji-derived LMP2 with GeneBank --- p.101Chapter 3.12 --- Isoelectric point analysis --- p.103Chapter Chapter 4 --- DiscussionsChapter 4.1 --- Overall strategy for the cloning and sequencing of EBV LMP 2 gene --- p.106Chapter 4.2 --- Implications of the results obtained in sequencing --- p.107Chapter 4.3 --- Results interpretation --- p.108Chapter 4.3.1 --- Cell culture --- p.108Chapter 4.3.2 --- Extraction of total RNA --- p.108Chapter 4.3.3 --- PCR amplification --- p.109Chapter 4.3.4 --- Confirmation of the PCR amplified cDNAs using nested PCR --- p.109Chapter 4.3.5 --- Confirmation of the PCR amplified cDNAs using restriction enzyme digestion --- p.110Chapter 4.3.6 --- Ligation of EBV LMP 2 cDNA to pGEM-T Easy Vector --- p.111Chapter 4.3.7 --- Transformation and screening --- p.114Chapter 4.3.8 --- Extraction of plasmid DNA and digestion with restriction enzyme --- p.115Chapter 4.4 --- DNA sequencing and sequence homology --- p.116Chapter 4.5 --- Amino acid sequence homology --- p.117Chapter 4.6 --- Hydrophobicity analysis --- p.119Chapter 4.7 --- Protein secondary structure analysis --- p.120Chapter 4.8 --- Isoelectric point analysis --- p.122Chapter 4.9 --- Summary of results --- p.122Chapter 4.10 --- Conclusions --- p.124References --- p.12

Elispot assay of HLA class I restricted EBV epitope choices in Hong Kong donors.

Xu Xuequn.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 100-125).Abstracts in English and Chinese.Chapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Epstein-Barr (EBV) Virus --- p.1Chapter 1.1.1 --- Virus Structure and Genome Structure --- p.1Chapter 1.1.2 --- Virus Types --- p.2Chapter 1.2 --- EBV Infection and malignancies --- p.3Chapter 1.2.1 --- In Vitro Infection --- p.3Chapter 1.2.2 --- Infection in the Natural Host --- p.8Chapter 1.2.3 --- Malignancies Associated with EBV --- p.11Chapter 1.3 --- T Cell-Mediated Immune Response to EBV --- p.16Chapter 1.3.1 --- The Pathway of Cell-Mediated Immune Response in Viral Infection --- p.16Chapter 1.3.2 --- Cell-Mediated Immune Response to EBV --- p.18Chapter 1.3.3 --- The Feature of CTLs Response to EBV --- p.20Chapter 1.4 --- CTLs to EBV Relevant MalignancieśؤApplications and Challenges --- p.21Chapter 1.5 --- HLA Polymorphisms and Strategy of Epitope-Based CTLs Therapy --- p.24Chapter 1.6 --- The Effect of HLA Polymorphism on EBV-Specific CTL Epitope Choice in Southern Chinese --- p.27Chapter 1.7 --- ELISPOT Assay 226}0ؤ Detection of CTLs Response --- p.32Chapter 1.8 --- Aim of This Study --- p.37Chapter Chapter 2: --- Material and Methods: --- p.39Chapter 2.1 --- Peptides --- p.39Chapter 2.2 --- PBMCs Preparations --- p.43Chapter 2.3 --- PBMC Counting and Cells Dilution --- p.43Chapter 2.4 --- Elispot Assay --- p.44Chapter 2.5 --- Counting the Spots --- p.45Chapter 2.6 --- Spots Forming Cells (SFC/106) and Positive Standard --- p.46Chapter Chapter 3: --- Results --- p.47Chapter 3.1 --- Validation of ELISPOT assay methodology --- p.47Chapter 3.2 --- CTLs Response to Each Epitope in the Population --- p.55Chapter 3.2.1 --- Positive Response to A11 Restricted and Mutant Epitopes in the Population --- p.55Chapter 3.2.2 --- Positive Frequencies of A2 Restricted Epitopes in the Population --- p.63Chapter 3.2.3 --- Positive Frequencies of Other HLA Allele Restriction Peptides --- p.70Chapter 3.3 --- CTLs Response Frequencies Categorized by Proteins --- p.74Chapter 3.3.1 --- "CTLs Response to LMP1, LMP2, EBNA1 Epitopes" --- p.74Chapter 3.3.2 --- "CTLs Response to EBNA2, EBNA-LP Epitopes, EBNA3 Epitopes" --- p.75Chapter 3.3.3 --- CTLs Response to LYTIC Epitopes --- p.79Chapter 3.4 --- Summary --- p.80Chapter Chapter 4: --- Discussion --- p.82Chapter 4.1 --- Discussion of A11 Restricted Epitopes --- p.82Chapter 4.2 --- Discussion of A2 Restricted Epitopes --- p.86Chapter 4.3 --- Discussion of Other HLA Restricted Epitopes --- p.89Chapter 4.4 --- "Discussion ofLMPl, LMP2, EBNA1 Epitopes" --- p.92Chapter 4.5 --- "Discussion of EBNA2, EBNA3, and EBNA-LP epitopes" --- p.96Chapter 4.6 --- Discussion of LYTIC Epitopes --- p.96Chapter 4.7 --- Discussion of Summary --- p.98Chapter Chapter 5 --- Conclusion --- p.99Chapter 6 --- Reference --- p.100Chapter 7 --- Appendix --- p.126Chapter 7.1 --- "Appendix 1, raw data of Elispot assay on CTLs response to EBV relevant epitopes m Hong Kong donors" --- p.126Chapter 7.2 --- "Appendix 2, frequencies from highest cell number wells of the peptides (SFC/106)" --- p.126Chapter 7.3 --- "Appendix 3, typical Elispot assay figure " --- p.12

The involvement of oncogenic DNA viruses in Hodgkin's disease

At the outset of this project Epstein-Barr virus (EBV) was associated with a proportion of cases of Hodgkin's disease (HD). Clonal EBV genomes were detected in HD tumour material however the localisation of EBV within the malignant cells had not been clearly demonstrated. Following the availability of monoclonal antibodies to the EBV latent genes, the expression of LMP-1 and EBNA-2 was investigated in a series of HD cases. EBV LMP-1 was expressed in Reed-Sternberg (RS) cells in the majority of cases studied, however there was a lack of expression of EBNA-2. The detection of LMP-1, which has been shown to have oncogenic potential, has strengthened the evidence that EBV is involved in the pathogenesis of a proportion of HD cases. These results indicated that a distinct pattern of EBV latent gene expression, designated Lat II, was observed in HD. As LMP-1 has been shown to upregulate a number of cellular genes, the expression of CD23 and bcl-2 in HD were examined. CD23 and bcl-2 were rarely detected in RS cells. There was no correlation between LMP-1 expression and the presence of CD23 or bcl-2. These results indicate that the role of EBV in HD is independent of the upregulation of CD23 and bcl-2. Further evidence that EBV is localised to RS cells in HD was obtained following the detection of EBER RNA in RS cells using an in situ hybridisation technique. Comparison of techniques to detect EBV in HD tumour material indicated that the EBER RNA in situ hybridisation assay was the most useful and reliable method of determining EBV latent infection. We have categorised HD cases which were EBV-positive by in situ assays or using Southern blot hybridisation for the detection of clonal EBV genomes as EBV-associated. Using tine above criteria the epidemiological features of HD were investigated with respect to EBV. Paediatric HD cases, in particular cases <10 years of age, older adults and cases of MCHD subtype were strongly EBV-associated. There was no evidence that EBV is a useful prognostic marker. Although the epidemiological features of HD suggest that young adult HD is most likely to be caused by an infectious agent our results indicated that these cases, in particular NSHD, were seldom EBV-positive. We have speculated that another virus may be involved in this age group. In order to eliminate the possibility that other known DNA viruses may be involved in HD, we examined clinical samples from HD, NHL and reactive conditions for the presence of adenovirus, SV40, LPV and HHV-7. We have not detected any of the viruses in these clinical samples. The implications of these results will be discussed further in this thesis