Genetic and functional diversity of central nervous system (CNS) derived Human Immunodeficiency Virus type 1 (HIV-1) tat from Tuberculous Meningitis (TBM) patients.

Abstract

INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (tat) is a regulatory gene that encodes the transactivator of transcription Tat protein. The Tat effectively increases the activity of the HIV-1 5’ long terminal repeat (5’ LTR) viral promoter to transcribe viral genes. The tat gene has two exons; the first 72 amino acids of Tat are encoded by the first exon, whilst amino acids 73 – 101 are encoded by the second exon. Exon 1 of Tat is sufficient for the transactivation of the 5’ LTR and therefore was the focus of this study. The Tat encoded by exon 1 consists of 5 functional domains these include: the acidic domain (domain I) comprising amino acids 1 – 21, this is a proline rich domain with high sequence variation; the cysteine-rich domain (domain II) comprising amino acids 22–37, is composed of 6 well conserved cysteine residues in subtype C Tat proteins, a mutation at any of the 6 cysteine residue results in loss of Tat activity; the core domain (domain III) comprising amino acids 38–48, is made of a hydrophobic motif and is relatively well conserved. Together, the first 48 amino acids of Tat comprising domains I – III, allow for the transactivation activity of Tat responsible for enhancing viral gene transcription. The basic domain (domain IV) is an RNA-binding domain made up of amino acids 49 – 57 which allows for the binding ability of Tat to the TAR loop structure of the 5’ LTR. Lastly the glutamine-rich domain (domain V) comprised of amino acids 58 – 72, also concentrated with basic amino acids, has the highest sequence variation in Tat. During the early stages of infection, HIV-1 enters the central nervous system (CNS) and replicates at marginal levels compared to high viral replication in the periphery. Yet, there is higher HIV-1 RNA levels in the in the cerebrospinal fluid (CSF) compared to plasma of tuberculosis meningitis (TBM) co-infected patients. However, the mechanisms driving the higher viral replication in the CNS of TBM patients are not well understood. Therefore, the major aim of this study is to characterise genetic and functional diversity of CNS and plasma derived Tat from TBM coinfected patients. We hypothesized that TBM coinfected patients will display genetically distinct HIV-1 tat variants in the CSF as a driver or consequence of higher viral replication in this compartment compared to plasma.

METHODS Viral RNA was extracted from matched CSF and plasma samples obtained from 20 HIV- 1 chronically infected patients (17 TBM and 3 non-TBM) using the QIAmp viral RNA Mini kit (Qiagen Inc., Valencia, CA, USA). Extracted viral RNA was reverse transcribed into viral DNA using SuperScript IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and amplified using two rounds of (nested) PCR with the Platinum ® Taq DNA Polymerase High Fidelity PCR kit (Thermo Fisher Scientific, Boston, MA, USA). Genetic diversity of plasma and CSF derived isolates was assessed in 19 patients (16 TBM and 3 non-TBM) by sequencing, neighbour-joining phylogenetic analysis and both interpatien and intrapatient diversity analysis. The Tat sequences with previously reported mutations that affect Tat function were selected for downstream functional assays. Twelve tat PCR amplicons were cloned into a pTargeT™ expression plasmid (Promega Corporation, Madison, WI). Recombinant pTargeT clones containing patient derived HIV-1 tat was propagated using the QIAfilter Plasmid Maxi Kit (Qiagen Inc., Valencia, CA, USA) to transfect the TZM-bl mammalian cells, which contains the luciferase gene luc under the control of the LTR promoter. A luciferase assay was done to measure the relative luminescence for each Tat mutant and this was correlated to markers of disease progression such as viral load. RESULTS The phylogenetic data from our study show that sequences from plasma and CSF derived HIV-1 tat clustered closely per patient. Genetic variation was seen as varying branch lengths between patient clusters. However, our data do not show significant nucleotide differences between the plasma and CSF tat sequences with a p-distance of 0.059 and 0.062 respectively (p = ns). Additionally, our data revealed that the amino acid sequences were the same between the CSF and plasma compartments, except in 5% of patients that showed differences in positions that were not previously reported to affect Tat activity. However, Tat diversity was observed to occur in all 5 domains of the first 72 amino acids of Tat namely: V4I, P21A, K24S, H29R, S31C, S46Y, R52W, S57R, P59S and D64G.

The functional data from our study revealed that most patient derived Tat mutations occurred in combination with other previously reported mutations. Interestingly, Tat mutations that occurred together with P21A in five different patients showed a showed strong positive correlation with CSF viral load in the CNS (p = 0.003; r = 0.98). CONCLUSION We reject our hypothesis that CNS specific Tat mutations were responsible for the high viral load in the CNS of patients who have TBM, as the allele frequencies of reported amino acid substitutions were represented in equal proportions within plasma and CSF derived Tat variants. Furthermore, our functional data shows that majority of all Tat variants from the TBM group had a reduced capacity to transactivate the 5’ LTR. Whilst we cannot confirm that Tat is responsible for the higher viral replication seen in the CNS of TBM coinfected patients, our data demonstrate that all Tat variants with a P21Anmutation significantly correlates to viral replication in the CNS. Future studies should perform site directed mutagenesis to determine the exact mutations that mediate LTR activity.