Abstract

MicroRNAs (miRNAs) are 17nt-24nt long non-coding RNAs which regulate gene expression by direct base pairing of the miRNA with target mRNAs in the 3’ UTR. This form of post-transcriptional regulation between miRNAs and their targets is quickly being recognized as a central regulatory mechanism necessary for most normal cellular functions. In fact, this regulation is essential from the development of the embryo to differentiation of numerous cell types, and from maintenance of health to the development of cancer and other diseases by dysregulated miRNAs. Understanding the complexities of miRNA-mRNA interactions at different stages of growth and development is therefore critical for delineating pathogenic pathways of various human diseases, and for the development of disease-specific therapeutics. ❧ Chapter One of this dissertation presents an introduction to miRNAs and a short synopsis of the major topics associated with miRNAs such as the history of miRNA discovery, miRNA biogenesis, mechanisms by which miRNAs regulate their targets, and the basic principles of miRNA-mRNA interactions. Also presented are short discussions on the concept of viral miRNAs and an explanation of the terminologies used such as miRNA and miRNA-like. ❧ The next chapter introduces our first report on the discovery of novel miRNA-like sequences in the genomes of several HIV-1 isolates and is presented exactly as published. The research presented in this thesis is based on Dr. Rasheed’s Laboratory’s research on the proteomics profiles of HIV-infection, which indicated that HIV-1 infection alone can dysregulate hundreds of proteins during chronic replication of HIV-1. Since microRNAs are directly involved in regulating protein expression, we proposed to search for viral miRNAs using in silico methods. Using metagenomics tools we were able to identify 8 cellular microRNAs which are predicted to bind and regulate the mRNAs of multiple proteins that were dysregulated by experimental HIV infection of CD4+ T-cells in vitro. Subsequent analysis of these miRNAs revealed that one cellular miRNA, miR-195, showed near perfect homology with five HIV-1 genomes from South Africa and four additional miRNAs showed significant homology with other African strains. We propose that these miRNA sequences may have evolved to self-regulate survival of the virus within its host by evading innate immune responses. ❧ In Chapter Three we take this idea further by examining all known human miRNA sequences, and comparing them to all known genomic and subgenomic HIV sequences contained in global databases (approximate n=400,000). By using the latest bioinformatics and high-throughput analyses, we examined hundreds of thousands of HIV sequences and compared them to all known human mature miRNA sequences. This examination resulted in the discovery of 15 mature human microRNAs within the protein-coding regions of 20 distinct HIV strains. While most of these miRNAs were clustered within the env region of HIV-1 genomes, several miRNAs were present in the gag, pol, nef, and LTR viral regions of multiple HIV-1 strains. Furthermore, most of these viral miRNA sequences exhibited 100% complete homology to the cellular miRNA counterparts and others had minor mutations. Of particular interest of this thesis is a key finding of the human miR-4644 in 34 distinct HIV isolates from different regions of the globe. ❧ Chapter Four represents an in-depth investigation into the nature of the miR-4644 and its matching HIV-1 isolates. These miR-4644 sequences were localized to the same exact position in each of the 34 strains, and that position is the boundary of the gp120-gp41 junction of the env gene. This viral region, which encodes a particular highly conserved amino acid motif, is also recognized as the binding site for furin, the cellular protease which catalyzes the eventual cleavage of gp160 into its active form. ❧ Another interesting twist on this discovery is the fact that miR-4644 is predicted to target the furin mRNA in silico. This means that the miR-4644 miRNA, embedded within the protein-coding region of the HIV-1 genome, may regulate the very enzyme that ultimately catalyzes proteolytic cleavage at precisely the same position, at the amino acid level. The implications are further discussed, and include the possibility that this viral miRNA may serve to regulate a critical maturation step in the infectivity of the HIV particle and may therefore utilize a new pathway in the modulation of HIV pathogenesis. ❧ In Chapter Five we examine another miRNA, miR-6763, more closely. Of the fifteen viral miRNAs that were discovered in relation to proteins that were dysregulated due to HIV-1 infection of T-cells, miR-6763 is especially interesting for two reasons: 1) the mature miRNA exhibits 100% complete homology across its entire length with its cellular miRNA counterpart, and; 2) this miRNA is present in three different and geographically diverse HIV-1 isolates. Upon further investigation, we have found that this miRNA lies in the 3’ LTR region of the HIV-1 viral genome and is co-located with a binding site for the transcription factor called specificity protein 1 (Sp1). Moreover, the Sp1 site is duplicated in all three HIV isolates. The two Sp1 sites are placed in tandem and together show 100% homology with miR-6763. ❧ The miR-6763 is also predicted to target and downregulate CD4 in silico. The significance of these findings is discussed in Chapter Six, which examines target sites in the 3’ UTR of the CD4 mRNA. The CD4 molecule is integral to the establishment of infection of HIV by providing a critical cell surface receptor for virus binding. In our investigation, we found that all three of our newly identified miRNAs that we have investigated in detail, mir-195, miR-4644, and miR-6763, are all predicted to downregulate the CD4 mRNA by binding to target sites in the CD4 3’UTR, presumably limiting expression of this molecule. An additional interesting note is that we have found that two putative HIV-encoded miRNAs, known as hiv1-miR-TAR-5p and hiv1-miR-H1, also have a predicted target site in the CD4 mRNA. ❧ Finally, we will discuss all novel findings discovered in this research and their implications on viral infection, replication, latency, pathogenesis and human disease. The possibility of a new regulatory pathway will be explored in the context of this new information. Our identification of new and novel viral miRNA molecules in the HIV-1 genome may serve to increase the knowledge of viral miRNA regulation and aid in the development of targeted therapies toward the assuagement of this global pandemic.