Abstract
Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.
Highlights
Human immunodeficiency virus (HIV) is the major causative agent of acquired immunodeficiency syndrome (AIDS) in humans
Several such simian immunodeficiency virus (SIV) backbones have been used for simian-human immunodeficiency virus (SHIV) generation, and in a recent finding, it was demonstrated that the backbone plays a minimal role in the degree of infectivity of SHIVs [11]
The main disadvantage of this conventional methodology for SHIV cloning is that it is dependent on the presence or generation of exclusive restriction sites
Summary
Human immunodeficiency virus (HIV) is the major causative agent of acquired immunodeficiency syndrome (AIDS) in humans. Since there are several limitations in obtaining human tissues/samples other than blood, investigators use non-human primates (NHPs) as models to study HIV infection and pathogenesis. Several isolates of the simian immunodeficiency virus (SIV) cause an AIDS-like disease in NHPs. Since HIV does not infect NHPs, a chimeric virus, the simian-human immunodeficiency virus (SHIV) has been engineered to contain HIV genes.
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