Advances in next-generation sequencing platforms during the past decade have resulted in exponential increases in biological data generation. Besides applications in determining the sequences of genomes and other DNA elements, these platforms have allowed the characterization of cell-wide mRNA pools under different conditions and in different tissues. In 2009, Ingolia and colleagues developed an extension of high-throughput sequencing that provides a snapshot of all cellular mRNA fragments protected by translating ribosomes, dubbed ribosome profiling. This approach allows detection of differential translation activity, annotation of novel protein coding sequences and variants, identification of ribosome pause sites and estimates of de novo protein synthesis. As with other sequencing based methodologies, a major challenge of ribosome profiling has been sorting, filtering and interpreting the gigabytes of data produced during the course of a typical experiment. In this thesis, I developed and applied computational pipelines to interrogate ribosome profiling data in relation to gene expression in several viruses and eukaryotic species, as well as to identify sites of ribosomal pausing and sites of non-canonical translation activity. Specifically, I applied various control analyses for characterizing the quality of profiling data and developed scripts for visualizing genome-based (exon-by-exon) rather than transcript-based ribosome footprint alignments. I also examined the challenge of mapping footprints to repetitive sequences in the genome and propose ways to mitigate the associated problems. I performed differential expression analyses on data from coronavirus-infected murine cells, retrovirus-infected human cells and temperature-stressed Arabidopsis thaliana plants. Dissection of translational responses in Arabidopsis thaliana during heat shock or cold shock revealed several groups of genes that were highly upregulated within 10 minutes of temperature challenge. Analysis of the branches of the unfolded protein and integrated stress responses during coronavirus infection allowed for deconvolution of transcriptional and translational contributions. During the course of these analyses, I identified errors in a recently publicized algorithm for detection of differential translation, and wrote corrections that have now been pulled into the repository for this package. Comparison of the translational kinetics of the dengue virus infection in mosquito and human cell lines revealed host-specific sites of ribosome pausing and RNA accumulation. Analysis of HIV profiling data revealed footprint peaks which were in agreement with previously proposed models of peptide or RNA mediated ribosome stalling. I also developed a simulation to identify transcripts that are prone to generating RPFs with multiple alignments during the read mapping process. Together, the scripts and pipelines developed during the course of this work will serve to expedite future analyses of ribosome profiling data, and the results will inform future studies of several important pathogens and temperature stress in plants.

Couverture)Les rétrovirus sont des virus enveloppés à ARN simple brin omniprésents dans le monde animal et sources de nombreuses pathologies. Les rétrovirus de vertébrés comprennent sept genres dont les gamma et deltarétrovirus qui sont l’objet de ces travaux. Les rétrovirus dits endogènes (ERV), par opposition à leurs homologues infectieux exogènes, sont présents dans les cellules germinales et font partie intégrante du patrimoine génétique, avec transmission mendélienne. Au cours de l'évolution, les ERV ont fait l'objet de mutations, rendant défectives la plupart des copies dans les génomes de vertébrés, avec quelques exceptions notoires. De fait, certaines copies maintiennent de larges cadres de lecture suite à une pression de sélection positive.Rétrovirus exogènes et ERV partagent une organisation génétique similaire. Leurs glycoprotéines d’enveloppe (Env), dont une des propriétés est de lier un récepteur cellulaire, comprennent une composante de surface (SU) associée à une partie transmembranaire (TM). La SU des Env γ et -rétrovirales porte un module RBD (Receptor-Binding Domain) qui lie un récepteur appartenant à la famille SLC (Solute Carriers) des transporteurs de nutriments. Les SLC présents à la surface cellulaire conditionnent le métabolisme des cellules. Afin de pallier l'absence d'anticorps fiables reconnaissant les parties extracellulaires (exofaciales) des SLC, le laboratoire a dérivé des RBD solubles comme ligands des SLC, permettant de suivre leur expression à la surface cellulaire et ainsi, évaluer le métabolisme cellulaire.Parmi les ERV, certaines env partiellement ou entièrement conservées jouent un rôle physiologique essentiel dans les organismes qui les portent. Une hypothèse de mon laboratoire d’accueil est l’existence de RBD endogènes de mammifères capables de moduler le métabolisme cellulaire de leurs hôtes. Dans ce contexte, mes travaux sont articulés autour de deux axes : (i) identifier et produire de nouveaux RBD dérivés des ERV et (ii) identifier de nouveaux transporteurs de type SLC reconnus par des RBD issus de rétrovirus exogènes et ERV de mammifères. Nous avons identifié et caractérisé deux nouveaux RBD humains endogènes (HERV-41 et HERV-89), entrés et conservés chez les primates de l’Ancien Monde il y a environ 35 millions d’années. Nous avons caractérisé leurs séquences PBS (Primer Binding Site), amorces putatives de la réplication rétrovirale, comme étant complémentaires de l’ARNtLeu ou ARNtArg pour HERV-89, et de l'ARNtGlu pour HERV-41. Les séquences env les plus proches dans le génome humain présentent respectivement 38% et 69% d'identité, indiquant l'appartenance de HERV-89 à deux nouvelles familles d'Env. Nous avons pu produire le RBD soluble de HERV-89, montrer que son récepteur est distinct de l'Env HERV ayant la séquence la plus homologue, et étudier sa distribution tissulaire. Le RBD HERV-89 lie un récepteur sur de nombreuses cellules souches et lignées cellulaires établies et nous avons montré par immunohistochimie que le récepteur est exprimé de manière différentielle dans les tissus humains sains et tumoraux. Parallèlement, nous avons dérivé une banque d'expression de 170 SLC que nous avons utilisée pour le criblage à haut-débit de récepteurs des Env gamma et deltarétrovirales. Cette banque nous a permis d'identifier le récepteur, longtemps recherché, de l’Env du virus de la leucémie bovine (BLV). De plus, en utilisant la transfection d'une banque d’expression d’ADNc dans des cellules de hamster, nous avons aussi identifié le récepteur du virus endogène félin ERV-DC14/FeLV-D comme étant le transporteur de cuivre et de cisplatine CTR1/SLC31A1.L’identification du récepteur de BLV pourrait notamment aider dans la lutte contre la transmission du virus et les pathologies associées qui affectent environ 5% du bétail infecté. De plus, les BLV-RBD et DC14-RBD constituent respectivement de nouveaux marqueurs et modulateurs potentiels du métabolisme, dont celui du cuivre / Retroviruses are enveloped, single-stranded RNA viruses, that are omnipresent in animals and the causal agents of a large array of pathologies. Vertebrate retroviruses are divided into seven genera, including the γ and -retroviral groups, which we study particularly. Endogenous retroviruses (ERV), as opposed to exogenous infectious viruses, are present in germline cells and as such are bona fide components of the host genome, with Mendelian transmission. Most ERV have been inactivated by purifying mutations during evolution, although a few copies have been subjected to positive selection pressure with conserved open reading frames (ORFs).Exogenous viruses and ERV that belong to gamma and deltaretroviruses share similar genetic organization and their envelope glycoproteins (Env) comprises a transmembrane (TM) and a surface (SU) component, which binds a specific receptor on the host cell membrane. The SU contains a receptor-binding domain (RBD), responsible for receptor recognition, while TM engages membrane fusion and harbors an immunosuppressive domain. Noticeably, some ERVs have maintained entire or partial ORFs in env, which have been shown, in certain cases, to have essential physiological functions.Another common feature of gamma and deltaretroviral Env is the nature of their receptors, which, when identified, all belong to the solute carrier family of nutrient transporters (SLCs). The laboratory derived soluble RBDs from complete Env that can bind cognate receptors and be used to monitor SLC receptor expression at the cell surface. This important property of RBDs overcomes the notorious lack of reliable anti-SLC exofacial antibodies and provides a new way to evaluate, or even modulate, cell metabolism.Our laboratory postulates that some endogenous RBD-coding genes have been positively selected in their hosts for properties linked to binding SLCs and modulating host cell metabolism. In this context, the aim of my work was to: (i) search for new natural endogenous RBDs and (ii) characterize SLC transporters recognized by RBDs derived from ERVs or exogenous infectious mammalian retroviruses.Here, we describe the identification of two novel human endogenous RBDs (HERV-41 and HERV-89), which each harbor a significant ORF. We estimated that both RBDs have been introduced into Old World primate genomes 35 MYA ago, after the separation with New World monkeys. HERV-89 and HERV-41 are included within retroviral elements that comprise potential primer binding sites (PBS) complementary to tRNALeu or tRNAArg, for HERV-89, and tRNAGlu, for HERV-41. The envs of HERV-89 and HERV-41 do not share more than 38% and 69% amino acid identity with the closest known HERVs, respectively, which indicates that they belong to two new Env families. We derived a soluble HERV-89 RBD and monitored its receptor cell and tissue distribution. Using the ligand by flow cytometry, we observed that a HERV-89 receptor is expressed in a large panel of established cell lines and stem cells. Immunohistochemistry on 94 healthy and tumor human tissue samples showed that HERV-89 receptor is largely distributed, with distinct expression patterns in healthy and tumor tissues. In parallel, we derived a 170 gene-containing SLC expression library for high throughput screening of SLC/ligand interactions. Using this partial human SLC library, we identified the long-sought receptor for bovine leukemia virus (BLV). Moreover, transfection of a cDNA library expression into hamster cells, led us to identify CTR1/SLC31A1, the copper and cisplatin transporter, as the receptor for the feline ERV-DC14/FeLV-D.As a ligand for the BLV receptor, BLV-RBD may be used to help controlling BLV transmission and prevent associated pathologies that affect 5% of infected cattle. Also, BLV-RBD and DC14-RBD can now be used as metabolic markers and modulators of their SLC cognate receptors, including copper metabolism, in the case of DC14-RBD.

Le génome des rétrovirus existe sous deux formes différentes : sous forme d'ARN simple brin, qui est traduit ou encapsidé, ou sous forme d'ADN double brin intégré dans le génome de la cellule hôte infectée. Cette dernière forme, l'ADN proviral, est indispensable à la production de tous les ARNm viraux nécessaires à la synthèse des protéines virales, qui en retour agissent sur la région promotrice située au niveau du LTR 5'. Cependant, l'ADN proviral possède un second LTR à son extrémité 3', capable de réguler une transcription antisens, orientée dans la direction opposée à celle contrôlée par le LTR 5'. L'ADN proviral a donc deux brins codants, ce qui offre au virus un plus grand potentiel de synthèse protéique. Dans le cas du Virus de l'immunodéficience Humaine de type 1 (VIH-1), la transcription antisens permet la production d'une protéine, appelée ASP (Antisense Protein). Dans ce manuscrit, nous démontrons que cette activité transcriptionnelle antisens s'exprime préférentiellement dans les cellules d'origine monocytaire, en particulier les cellules dendritiques ; une localisation membranaire de la protéine ASP a par ailleurs été mise en évidence dans ce type cellulaire. Nos résultats suggèrent également que la transcription antisens du VIH-1 est indépendante de la protéine Tat, et que par ailleurs les deux types de transcriptions ne sont pas exprimés simultanément au sein d'une même cellule. En outre, nos données soulignent que la séquence codante de la protéine ASP est très fortement conservée parmi les différents isolats viraux. Sur la base de l'ensemble de ces résultats, notre hypothèse est que la protéine ASP du VIH-1 possède des fonctions cruciales dans le cycle réplicatif des rétrovirus, indépendantes de la production virale. / Genome of retroviruses exists in two different forms: as single-stranded RNA that is translated or packaged, or as double-stranded DNA integrated into the genome of the infected host cell. The latter form, the proviral DNA, is essential for the production of all viral mRNAs required for the synthesis of viral proteins, which in turn act on the promoter region located at the 5 '-LTR. However, the proviral DNA has a second LTR at its 3 '-end, capable of regulating antisense transcription oriented in the opposite direction to that controlled by the 5'-LTR. The proviral DNA has then two coding strands, which gives the virus a greater potential for protein synthesis. In the case of the Human Immunodeficiency Virus type 1 (HIV-1), antisense transcription allows the production of a protein called ASP (Antisense Protein). In this manuscript, we demonstrate that this antisense transcriptional activity is preferentially expressed in cells of the monocyte lineage, in particular dendritic cells; a membrane localization of the ASP protein was also observed in this cell type. Our results also suggest that the antisense transcription of HIV-1 is Tat-independent, and what's more that the two types of transcription are not expressed simultaneously within the same cell. In addition, our data highlight that the ASP protein coding sequence is highly conserved among different viral isolates. Based on these results, our hypothesis is that the ASP protein of HIV-1 has critical functions in the replicative cycle of retroviruses, distinct from viral production.

L’intégrase (IN) du VIH-1 est une enzyme clé catalysant l’insertion de l’ADN proviral dans le génome cellulaire au sein d’un complexe nucléoprotéique d’intégration.Les nombreux efforts apportés à l’étude du mécanisme d’intégration ont permis d’accumuler de multiples données sur ce processus complexe. Cependant plusieurs questions essentielles demeurent sans réponses en particulier concernant l’architecture fonctionnelle des complexes d’intégration de VIH-1. En effet même si les complexes actifs pour l’intégration sont relativement bien définis leur chronologie précoce de formation demeure inconnue. De même les phases tardives de leur association au substrat naturel d’intégration que constitue la chromatine restent floues. Enfin le rôle des facteurs du complexe de préintégration (CPI) dans la régulation des ces mécanismes doit être déterminé.Mon travail de thèse s’est reposé sur trois axes s’articulant autour de ces questions:-Comment est régulée la dynamique des phases précoces d’attachement des oligomères d’intégrase sur les extrémités virales ?-Quel est l’impact de la structure de l’ADN cible et de la chromatine sur l’association active des complexes d’intégration ?-Quel(s) rôle(s) joue(nt) les facteurs du CPI dans ces phases ? / HIV-1 integrase (IN) is a key enzyme catalyzing the proviral DNA insertion into the cellular genome within a nucleoprotein integration complex.The numerous efforts made on the mechanism of integration have led to the accumulation of substantial data on this process. However many important questions are still open particularly on the functional architecture of the HIV-1 integration complexes. Indeed even if the active complexes involved in the catalysis of the integration are well described, the chronology of their early formation is still unknown. Moreover, the late association of these complexes with the host chromatin is also obscure. Finally the involvement of the IN cofactors inside the preintegration complex on these steps has to be elucidated.The project of my PhD relied on three axis articulated on these questions:-What is the dynamic of the IN oligomers association on the DNA viral ends?-What is the impact of the target DNA chromatin structure on the functional association of the integration complexes?-Involvement of the PIC factors on these steps?

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22333号 / 医博第4574号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 萩原 正敏, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Les rétrovirus sont des virus à ARN, enveloppés présents dans de nombreuses espèces animales de rente, chez les animaux de compagnie et chez l’homme. Une des particularités des rétrovirus concerne l’intégration du génome viral au sein du génome de la cellule infectée; cette intégration est réalisée par une enzyme virale, l’intégrase. Le projet de cette thèse vise à mieux comprendre le fonctionnement de cette enzyme notamment en identifiant des facteurs cellulaires interagissant avec celle-ci, facteurs qui pourraient être des agents favorisant le processus intégratif ou, au contraire, des agents restrictifs. Les intégrases de deux modèles de rétrovirus ont été utilisées dans cette étude : L’intégrase de RAV1, un rétrovirus exogène aviaire du genre des alpharétrovirus appartenant au sous-groupe A de la famille des ASLV. Cette enzyme virale est largement étudiée soit au niveau structural ou fonctionnel, mais les données concernant ses partenaires cellulaires sont rares et insuffisantes. La seconde intégrase est celle du PERV A/C, un rétrovirus endogène porcin du genre gammarétrovirus. Aucune information sur cette enzyme n’a été décrite jusqu’à présent. Ces deux enzymes, en fusion avec une étiquette 6xHistidine, ont été donc produites en bactérie, et en cellules d’insecte puis purifiées sur colonne d’affinité en FPLC. Leurs activités catalytiques ont été testées in vitro. Ces tests permettent de valoriser la capacité de l’intégrase à exercer principalement les 2 fonctions dont elle est responsable in vivo, le clivage en 3’ et le transfert de brins, et une activité qu’elle exerce exclusivement in vitro, la désintégration. Les protéines pures et actives ont ensuite servies à la vérification de leur interaction avec une protéine cellulaire, Brd2. La technique ‘Far western blot’ a ainsi permis de valider l’interaction entre l’intégrase de PERV et la protéine cellulaire, puis d’identifier les domaines de l’intégrase et de Brd2 impliqués dans cette interaction. A terme, l’identification de ce facteur cellulaire et la validation de son rôle dans le processus intégratif permettront de mieux comprendre ce processus particulier développé par les rétrovirus et pourront conduire au développement d’inhibiteurs dirigés contre cette interaction / A critical step for retroviral replication is the stable integration of the provirus genome into the genome of its host; this integration is realized by a viral enzyme, the integrase. The aim of this work was to better understand the functioning of the integrase, particularly, by identifying host factors that might interact with it, and which could be factors favoring the integration process or, restrictive factors. Therefore, we used two models of retroviral integrases: The integrase of RAV1, an alpharetrovirus belonging to the subgroup A of the family of ALSV. Although this viral enzyme is widely studied, still not enough data are available about its cellular cofactors. The second enzyme studied here is the integrase of PERV, a gammaretrovirus. No studies of either PERV integrase activities in vitro or of proteins interacting with this viral enzyme have been available until now. In the present study, we have expressed the PERV and ALSV integrases as fusion proteins with a 6xHistidine Tag in both Escherichia coli and insect SF9 cells. After that, we analysed their ability to mediate catalytic activities (3’-end processing, strand transfer and disintegration) in vitro. We also investigated the interaction of these two viral enzymes with the cellular protein Brd2, using the Far western blot method. Our results validate Brd2 as a cofactor of PERV integrase and point to the important role of particular domains of the PERV integrase and Brd2 in mediating the interaction. Finally, this study contibute to a better understanding of the precise interaction between cellular proteins and integrase, and may lead in the future to the development of protein-protein interaction inhibitors

Over three decades have passed since the discovery of HIV-1, yet an AIDS vaccine remains elusive. The envelope glycoprotein of HIV-1 gp120, is the most exposed protein on the viral surface and thus serves as an important target for vaccine design. However, various factors like high mutability of gp120, extensive glycosylation and very high conformational flexibility of gp120 have confounded all efforts to design a suitable immunogen that elicits broad and potent neutralizing antibodies against HIV-1. In Chapter 1, a brief description of the structural organization of HIV-1 along with the progress made and the difficulties encountered in the development of a vaccine are presented. In Chapter 2, the design and characterization of an outer domain immunogen of HIV-1 gp120 is discussed. The outer domain (OD) of the envelope glycoprotein gp120 is an important target for vaccine design since it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD based immunogens in the past have met with little success. In this work, we designed an OD immunogen based on the sequence of the HXBc2 strain, expressed and purified it from E. coli (ODEC). The ODEC molecule lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120 to increase solubility. Biophysical studies showed that ODEC is folded and protease resistant while ODEC lacking the designed mutations is highly aggregation prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody b12 with micromolar affinities, but not the non-neutralizing antibodies b6 and F105. Further improvement in the refolding protocol yielded a better structured molecule that bound CD4, b12 and VRC01 with sub-micromolar affinities. In rabbit immunization studies with animals primed with ODEC and boosted with gp120, the sera are able to neutralize Tier I viruses and some Tier II viruses like JRFL and RHPA with measurable IC50s. This is one of the first examples of a gp120 fragment based immunogen which was able to elicit sera that showed modest neutralization of some Tier II viruses. Subsequently amide hydrogen-deuterium exchange studies of ODEC showed that though the molecule is well-folded, it is labile to exchange. This might indicate why ODEC does not elicit high amounts of neutralizing antibodies. In Chapter 3, we report the design and characterization of two smaller fragments of gp120 (b121a and b122a) to target the epitope of the broadly neutralizing antibody b12. The region chosen comprised of a compact beta barrel in the lower part of the outer domain of gp120. Unlike ODEC, the fragments corresponding to these constructs were not contiguous stretches in gp120. Thus we used linkers to connect them. Further, nine designed mutations were introduced at exposed hydrophobic regions of the fragment to increase its solubility. The designed protein fragments were expressed in E. coli in order to prevent glycosylation and consequent epitope masking that might occur if expressed in an eukaryotic expression system. Biophysical studies showed that b121a/b122a are partially folded. Disulfide mapping studies showed that the expected disulfide bridges were formed. The designed immunogens could bind b12, but not the non-neutralizing antibody b6. Sera from rabbits primed with b121a/b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity against a 20 virus panel including Tier2 and 3 viruses such as PVO4, CAAN, CAP45 and ZM233. Sera from animals that received only gp120 showed substantially decreased breadth and potency. Serum depletion studies confirmed that neutralization was gp120 directed and that a substantial fraction of it was mediated by CD4 binding site (CD4bs) antibodies. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals, despite the restricted germline VH gene usage observed so far in broadly neutralizing CD4bs directed antibodies in humans. In Chapter 3, we also discuss design of a new construct b122d, which includes regions corresponding to b121a, but with linker connectivities similar to b122a. It was found to bind b12 with sub-micromolar affinity and also showed proteolytic resistance comparable to b121a. This indicated that though b121a showed better proteolytic resistance than b122a, it bound b12 poorly because one of the linkers might sterically occlude the b12 binding site. As the b12 binding site constructs based on the subtype B HXBc2 sequence elicited neutralizing antibodies, we chose to design similar constructs based on a subtype C sequence. The proteins (Cb122a and Cb122d) were purified from E. coli, characterized and found to bind b12 with micromolar affinity. The new constructs (b122d, Cb122a, Cb122d) will shortly be tested in animal immunizations. Disulfides are known to stabilize proteins by reducing the entropy of the unfolded state. In Chapter 4, we attempted to stabilize b122a by engineering disulfides. The disulfides are expected to rigidify the molecule and possibly improve its ability to elicit neutralizing antibodies. Some of the disulfides tested in b122a were predicted based on stereo-chemical criteria by the program MODIP (Modeling Disulfide Bridges in Proteins), while others were chosen at non-hydrogen bonded positions (NHB) on anti-parallel beta strands, based on earlier studies in the lab. Some of the disulfide mutants showed better binding to b12 and increased protection to enzymatic digestion. These disulfides were subsequently engineered into other b12 binding site constructs, namely b122d, Cb122a and Cb122d and these were biophysically characterized. Amongst the various disulfides that were tested in b122a, the one at 293-448 (according to HxBc2 numbering) was found to improve the binding to b12 by about ~16-fold. Not only did this disulfide improve the binding of b122a to b12, it also showed similar improvement in case of b122d and both the subtype C constructs tested. Moreover, since the position 293-448 is an exposed NHB position of an anti-parallel beta strand, spontaneous formation of the disulfide and the improved binding to b12 for all the proteins tested reinforces the fact that cysteines engineered at such positions leads to formation of a stabilizing disulfide. All the proteins containing the 293-448 disulfide will be used in future for rabbit immunization studies to examine if they elicit better neutralizing antibodies than the parent b122a molecule. As discussed in Chapter 2, ODEC showed a very fast rate of hydrogen exchange, indicating that it is flexible. As the 293-448 disulfide improved the binding of b12 binding site constructs, in Chapter 5, disulfides at exposed NHB positions were introduced in the context of ODEC. Previously engineered inter-domain disulfides have been shown to reduce the conformational flexibility of gp120. The disulfides in the lower beta barrel of the outer domain which harbors the CD4 binding site were found to be monomeric, oxidized and could bind neutralizing CD4bs antibodies better than the WT protein. On the other hand, the disulfides in the upper barrel of the outer domain were aggregated and bound antibodies poorly compared to the WT protein, indicating that this part of the molecule may not be well structured in the fragment. However, there was no significant change in the hydrogen exchange kinetics for these mutants. Mutations in the Phe-43 cavity of gp120 (S375W/T257S) which constrain gp120 in the CD4 bound conformation were also tested in ODEC (ODEC-CF). This protein was found to bind CD4 and VRC01 about 8 and 2 times better respectively than WT ODEC. These improved immunogens will be used shortly in rabbit immunization studies. In an attempt to improve the immunogenicity of the gp120 fragment proteins, b121a, b122a and ODEC were displayed on/conjugated to the surface of Qβ virus like particles in Chapter 6. Exposed single cysteine mutants of these proteins were purified, characterized biophysically and found to have the single cysteine free for conjugation. These were subsequently conjugated to the Qβ virus like particles through click chemistry (carried out in Prof. MG Finn’s lab at TSRI), purified and used for rabbit immunization studies. The gp120 ELISA titers of the elicited sera showed that conjugation may be a better option to display foreign antigens on the surface than genetic fusion. There was no difference in the ELISA titers with and without adjuvant, indicating that the particles are sufficiently immunogenic in themselves. Sera from these studies will be tested in neutralization assays. The overall utility of the particle based display approach will be assessed by comparing neutralization data from particle based immunizations to identical immunizations with unconjugated immunogens. Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the Env surface protein. Native Env consists of a trimer of gp120:gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs) directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. To understand this further, in Chapter 7, we have designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1/V2 loop. h-CMP-V1cyc is one such variant where 153 and 142 are the N and C terminal residues of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity relative to wtgp120. It binds the non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1 bound the V1V2 directed broadly neutralizing antibodies PG9/PG16 with ~20 fold higher affinity compared to wild type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support Env models in which the V1V2 loops are proximal to the trimer interface. In Appendix A1, peptide analogs of selected secondary structural elements of gp120 were designed. Some of them were grafted on known scaffold proteins. The synthesized peptides were characterized biophysically. Most of the peptides did not have a well-defined secondary structure, indicating that they are not stable in isolation. Hence they were not pursued for further studies. One helical peptide adopted a significant amount of structure in aqueous buffer and will be shortly conjugated to carrier proteins and used in immunization studies. In Appendix A2, we created error-prone PCR libraries and loop-randomization libraries of b12 binding site constructs and attempted to screen these for better b12 binding using phage-display. However the screening was unsuccessful as the phages showed non-specific binding to b12 antibody. These libraries will be screened in future using yeast display.