Amplification and Nucleotide Sequencing of Nef Gene of HIV

Abstract

Human Immunodeficiency Virus (HIV) is a single-stranded RNA virus that belongs to the Retroviridae family and the genus Lentivirus. HIV is the primary cause of acquired immunodeficiency syndrome (AIDS). The World Health Organization (WHO) reported almost 39 million cases infected with HIV worldwide with estimated 210,000 cases in Pakistan. Nef is an important accessory gene of HIV which encodes Nef protein. The Nef (negative regulatory factor) protein has three important roles during HIV infection. It down-regulates the cell surface receptors such as MHC molecules and CD4, it facilitates cell signaling, and increases viral particle infectivity by crossing all cellular barriers. In the current investigation, blood samples were collected from suspected patients from Lahore, Pakistan. Those HIV positive samples were included in the study that were confirmed through Real-Time Polymerase Chain Reaction and had viral load >100000 copies/mL. Those samples were then processed for Nef gene amplification, followed by Sanger nucleotide sequencing. Phylogenetic analysis was carried out utilizing the Neighbor Joining method followed by the Likelihood model of Maximum Composite. The amplified Nef gene sequence of our isolate QAU-AS1 grouped with a previously published sequence from Nigeria (Accession no. MT222940). Moreover, amino acid substitutions were also identified in Nef protein. The positions of these substitutions were analyzed with reference sequence. The 2D and 3D structures of query isolate and reference protein showed no significant change in its structural part, hence it may have limited role in overall protein structure. Further research is required to explore Nef gene of viral isolates from Pakistani patients to analyze possible mutations that have important role in pathogenicity. Keywords: HIV; Nef gene; Nef protein; Phylogenetic analysis; Mutational analysis