Abstract
HIV-1 has caused 35 million deaths globally, and approximately the same number is currently living with HIV-1. The trans-activator of transcription (Tat) protein of HIV-1 plays an important regulatory function in the virus life cycle, responsible for regulating the reverse transcription of the viral genome RNA. Tat is found in the nucleus of infected cells, but can also invade uninfected neighbouring cells. Regions within Tat responsible for these cellular localisations are overlapping and include a nuclear localisation signal (NLS) spanning 48GRKKRR, and a cell penetrating peptide (CPP) signal spanning 48GRKKRRQRRRAPQN. However, the mechanism by which this NLS/CPP region mediates interaction with the nuclear import receptors remains to be resolved structurally. Here, we establish that the HIV-1 Tat:NLS/CPP is able to form a stable and direct interaction with the classical nuclear import receptor importin-α and using x-ray crystallography, we have determined the molecular interface and binding determinants to a resolution of 2.0 Å. We show for the first time that the interface is the same as host factors such as Ku70 and Ku80, rather than other virus proteins such as Ebola VP24 that bind on the outer surface of importin-α.
Highlights
Tat is a transcriptional trans-activator and plays an important role during HIV-1 replication by binding to a short-stem loop structure, known as the transactivation response element (TAR) located at the 5′ end of HIV RNAs
The Tat:nuclear localisation signal (NLS)/cell penetrating peptide (CPP) region forms a direct interaction with importin-α
The NLS/CPP region of Tat, spanning residues 49–61, have been shown to contain a functional NLS, there has been recent debate as to whether the highly basic cell penetrating peptide region is bound using the importin-α adapter, or can bind directly to importin-β. Since this region contains a large stretch of positively charged residues, many of which of which could fit the definition of a classical NLS binding to importin-α, or an Arg rich importin-β interaction, we tested binding against both types of receptors
Summary
Tat is a transcriptional trans-activator and plays an important role during HIV-1 replication by binding to a short-stem loop structure, known as the transactivation response element (TAR) located at the 5′ end of HIV RNAs. Tat has been shown to localise to the nucleus in many studies, the mechanism by which it interacts with the nuclear import receptors has not been elucidated structurally[4, 5]. In vitro studies have shown that Tat is able to bind nuclear import receptors which mediate nuclear localisation[5, 15], a structural basis for this interaction remains to be elucidated. To determine the precise binding determinants that mediate interaction between the nuclear import receptor and Tat, the entire cell penetrating region of HIV-1 Tat, 48GRKKRRQRRRAPQN61, was recombinantly expressed as a GST-fusion and tested for binding to both importin-α and importin-β6, 16. Together with structural elucidation of the interface by x-ray crystallography, this study provides new insights into the interface between these two proteins which mediate localisation of Tat to the nucleus
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