Researchers have recreated the first steps of infection by HIV in a test tube, providing an up-close access to the virus.
HIV, also known as human immunodeficiency virus, is an enveloped virus with two copies of positive-sense single-stranded RNA. In order to infect host cells, HIV must reverse transcribe its single-stranded RNA genome into a double-stranded DNA copy. The virus must then integrate that copy into a host chromosome. Both of these processes are key targets for successful HIV-1 antiretroviral therapies. Additionally, while researchers have characterised these events separately, until now, they have not been reconstituted together outside of the cell. This is because viral core particles perform these processes deep within the infected cell cytoplasm and nucleus.
In a study, published in Science, researchers reconstituted processes of reverse transcription and integration in a cell-free system. The team used purified HIV-1 virions as the source of viral genomes and enzymes.
By gently permeabilising the viral membrane, the team were able to recapitulate the sequential processes of endogenous reverse transcription and integration from viral core particles. The team initiated reverse transcription with the addition of deoxynucleotide triphosphates. The DNA products appeared in high yields and in expected temporal order (early, intermediate and late transcripts). Provided that researchers added cell extract to the reaction mix, they could recapitulate integration of the resulting viral DNA into an exogenous target DNA. The researchers also performed deep sequencing and cloning to confirm that the viral DNA was integrated in a concerted fashion.
Most importantly, the team also found that these processes required the presence of capsid lattices of appropriate stability and geometry. They discovered that when they destabilised the capsid, HIV could no longer effectively replicate. This highlighted that the capsid is actually an essential component of the HIV infection process, rather than just for packaging.
The team anticipate that this cell-free system will enable systematic analyses of the key steps in viral replication and integration. This in turn could help improve drug design.
Image credit: Image Team – canva.com