STUDIES OF THE DIMERIZATION AND PACKAGING SIGNALS IN HIV-2 RNA

The investigation of sequences and structures in the 5\u27 untranslated leader region (5\u27UTR) of HIV genomic RNA is essential for understanding viral replication because the 5\u27UTR regulates several essential functions by the alternate presentation and sequestration of signals through conformational changes. Our main focus in this study was to understand those sequences and structures that are involved in dimerization and packaging of HIV-2 RNA. Progressing from previous findings, we studied in detail a 10-nucleotide palindrome sequence (pal; 5\u27-GGAGUGCUCC-3\u27) of the 5\u27UTR, located within the major packaging signal, upstream of the dimerization signal (SL1: stem loop-1). Pal has been shown to interact intramolecularly with SL1 to inhibit SL1-mediated HIV-2 leader RNA dimerization in vitro. We carried out three lines of experiments in an effort to understand the roles of pal in viral RNA dimerization and packaging. The first study was achieved through randomization of pal and the subsequent selection from a population of random-pal RNAs those with enhanced or diminished dimerization properties in vitro. We showed that the 3\u27-pal motif (3\u27-pal; GCUCC-3\u27) is involved in intramolecular interactions with a sequence downstream of SL1 that regulates SL1-mediated HIV-2 leader RNA dimerization. The second study was designed to investigate the role of 5\u27-pal motif (5\u27-GGAGU) in RNA packaging in vivo. Our findings indicated that the 5\u27-pal is essential for viral replication and genomic RNA packaging. Based on these findings, we proposed that 5\u27-pal is a binding element for the packaging proteins. Therefore, a third study was designed in which HIV-2 packaging proteins (Gag and NC) were expressed, purified, and assayed for binding with wild-type and mutant 5\u27-pal RNAs in vitro. These results suggested that the 5\u27-pal is a binding element for Gag protein. In summary, we have showed that pal is an important regulator of dimerization and packaging processes of HIV-2 RNA. We also demonstrated that pal is composed of two motifs with distinct functions. Overall, our study significantly contributes to the understanding of HIV-2 RNA dimerization and packaging, which may ultimately lead to the identification of novel antiretroviral targets

New windows into retroviral RNA structures.

BACKGROUND: The multiple roles of both viral and cellular RNAs have become increasingly apparent in recent years, and techniques to model them have become significantly more powerful, enabling faster and more accurate visualization of RNA structures. MAIN BODY: Techniques such as SHAPE (selective 2'OH acylation analysed by primer extension) have revolutionized the field, and have been used to examine RNAs belonging to many and diverse retroviruses. Secondary structure probing reagents such as these have been aided by the development of faster methods of analysis either via capillary or next-generation sequencing, allowing the analysis of entire genomes, and of retroviral RNA structures within virions. Techniques to model the three-dimensional structures of these large RNAs have also recently developed. CONCLUSIONS: The flexibility of retroviral RNAs, both structural and functional, is clear from the results of these new experimental techniques. Retroviral RNA structures and structural changes control many stages of the lifecycle, and both the RNA structures themselves and their interactions with ligands are potential new drug targets. In addition, our growing understanding of retroviral RNA structures is aiding our knowledge of cellular RNA form and function

Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?

During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets

In vitro selection and characterisation of human anti-HIV-1 antibody fragments

Generation of neutralising antibodies with broad specificity would be one of the effective approaches to control HIV-1 spread. It is clear that a method that allows rapid generation of neutralising antibodies is needed. This project aims at developing a novel approach to rapidly access human anti-HIV-1 antibodies in vitro by using ribosome display and selection from DNA libraries of HIV-1 patients. Two single-chain antibody libraries (M325 and K530) were constructed from two HIV-1 long-term non-progressors, whose sera showed cross-neutralising activities against various HIV-1 strains across a range of clades. In each library, total RNA was extracted from blood of each donor and used to synthesise cDNA. Families of 4 κ light chains, 9 λ light chains and 8 heavy chains were generated by using RT-PCR amplification. These fragments were then assembled with all possible combinatorial pairs to form diversified repertories in the form of VL-link-VH-partial CH. Both libraries were subjected to ribosome display for in vitro selection of functional antibodies. Ribosome display is a cell-free technique used to generate proteins that can bind to an immobilised antigen. During this process, the translated proteins are associated with their mRNAs, enabling a simultaneous selection of functional proteins and their gene. The employment of ribosome display facilitated rapid screening of two large libraries against recombinant gp120 (generated from patient K530)

The role of envelope glycoprotein in human immunodeficiency virus type 2 disease

Progression to AIDS following HIV-2 infection is generally slower than for HIV-1, however, rapid progression has been observed in some HIV-2 infected individuals. This disparity between HIV-1 and HIV-2 infection may be due to either HIV-2 having an attenuated viral phenotype or, alternatively, enhanced immunological control of HIV-2 replication, which may prolong the asymptomatic phase of disease. The envelope glycoprotein of HIV contributes to both of these phenomena; it contains regions that determine viral phenotype and is also a major target for host immunological responses. This thesis describes the development of a highly efficient plasmid vector for the PCR cloning and eukaryotic expression of recombinant envelope glycoproteins, together with an ELISA to measure the properties of these glycoproteins. These techniques have enabled us to perform quantification and functional analyses of various HIV-2 env glycoproteins (rgp105) and to examine their function, antigenicity and role in determining disease progression rate. Significant diversity in antigenicity and CD4 binding was found between clones derived from single isolates, between different isolates and between patients. However, we found no strong correlation between rgp105-CD4 affinity and viral phenotype or patient clinical status. Analysis of humoral responses to whole HIV-2 antigens and to rgp105 showed no difference between progressing and non-progressing patients. In contrast, fine mapping of the HIV-2 envelope using a panel of 210 overlapping 12mer oligopeptides identified two peptides within the gp36 transmembrane domain which were differentially recognised. Further investigation showed that humoral responses to amino acids 645-656 were significantly elevated in patients with better disease prognosis. Therefore, host humoral responses may play an important role in protecting the host from disease and in extending the clinically asymptomatic phase of HIV-2 infection. These findings maybe important in the development of preventive or prophylactic vaccines for the treatment of both HIV-1 and HIV-2 infections

Synonymous genome recoding : a tool to explore microbial biology and new therapeutic strategies

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies

Mechanistic understanding of \u3ci\u3eN\u3c/i\u3e-glycosylation in Ebola virus glycoprotein maturation and function

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1 and the fusion-mediating GP2 subunits and incorporated into virions to initiate infection. GP1 and GP2 form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1 NGSs are not critical, the two GP2 NGSs, Asn563 and Asn618, are essential for GP function. Further analysis uncovered that Asn563 and Asn618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that bothCNXand CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn563 and Asn618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality

Characterisation of llama antibody fragments able to act as HIV-1 entry inhibitors

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the functional envelope spike, which consists of trimers of gp120 bound to gp41. Most variants of HIV-1 enter cells through attachment of the envelope spike to the main cellular receptor CD4, allowing interaction with a co-receptor and eventually fusion of viral and cellular membranes. Neutralising antibodies inhibit HIV-1 entry by targeting epitopes on the functional spike. HIV-1 has, however, evolved several ways to evade recognition by antibodies, including variable regions, carbohydrates, and conformational masking. As a result, the neutralising antibody response in HIV-1 infection and post-immunisation is generally narrow, and only a handful of broadly neutralising monoclonal antibodies have been reported. In this thesis, the isolation and characterisation of novel, broadly neutralising antibody fragments derived from llamas is described. Llamas produce antibodies devoid of light chains, which have their antigen-binding properties confined to a single fragment, the VHH, and a preference for cleftrecognition. VHH were isolated from llamas immunised with recombinant gp120 using phage display-based methods. In order increase the chances of isolating neutralising VHH, a functional selection strategy was employed, involving a competitive elution with soluble CD4. Three VHH able to neutralise HIV-1 primary isolates of subtype B and C were characterised. These VHH bound to gp120 with high affinities and competed with soluble CD4 and antibodies to the CD4-binding site for this binding, indicating that their mechanism of neutralisation involves interacting with the functional envelope spike prior to binding to CD4. These results indicate that llama VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for HIV-1 microbicide development. Anti-gp120 VHH might also prove useful in defining neutralising and non-neutralising epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design