Despite the availability of antiretroviral therapy (ART), the human immunodeficiency virus (HIV) is still regarded as a pandemic in the twenty-first century, with an estimated 39 million people living with HIV (PLWH), 1.3 million new HIV infections, and 630 000 deaths from AIDS documented in United Nations Programme on HIV/AIDS (UNAIDS) 2022 report. Normally, HIV-1 viral load is marginally lower in the cerebral spinal fluid (CSF) than in the plasma of ART-naïve PLWH. Paradoxically, there is a higher viral load in the CSF than plasma of ART-naïve individuals co-infected with tuberculous meningitis (TBM). However, the mechanisms that govern high viral load in the CSF compared to plasma of TBM co-infected ART naïve individuals remain to be determined. Hence, the current study aims to investigate the effect of Sp1IIIT5A mutation alone or in combination with other mutations on the ability of Sp1 site III to bind Sp1 transcription factor and mediate LTR transcription activity. The gene block containing Sp1III5A was cloned into the pGL3 plasmid and sequenced to confirm the mutation. The recombinant PGL3-LTR was transfected into Jurkat and astrocyte cells to assess transcription activity. The Sp1III5A mutation was successfully introduced, and it significantly exhibits high transcription activity in Jurkat and astrocyte cells. The presence of a single-nucleotide variation (Sp1III5A) in the Sp1 site III of the LTR sequence of HIV-1C increases the transcription activity in TBM co-infected individuals.