論文の公開元へCleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion
概要
Introduction:
Type-I interferons (IFN-I) are the innate immune system’s principal defense against viral infections. Almost all cell types are capable of producing IFN-I upon recognition of foreign molecular patterns of viruses through pattern recognition receptors (Ni et al., 2018). The TRAF Family Member Associated NFKB Activator protein binding kinase-1 (TBK1) is one of the most important cellular protein involved in the IFN-I secretion pathway. (Beachboard and Horner, 2016). Failure to mount an effective IFN response leads to widespread systemic infection in the host (Huang et al., 2019).
Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host’s innate immunity to survive and replicate to sustain infection. Suppression of IFN-I production is one among the multiple escape strategies used by HIV-1 to prevent its clearance (Guha and Ayyavoo, 2013). HIV-1 protease (HIV-1 PR), the viral protein which helps in viral maturation is one of the important targets of the potent antiretroviral therapy (ART) drugs; protease inhibitors (PI). Apart from its de novo action in viral replication, HIV-1 PR has also been observed to cleave host various intracellular proteins of the host cell (Yang et al., 2012).
We hypothesized that HIV-1 PR could cleave TBK1 and reduce IFN-I production to favor HIV-1 replication. We also hypothesized that this HIV-1 PR mediated immune evasion mechanism could be attenuated by PIs.
Methods:
In this study we perform a comprehensive screening of a library of 412 human kinases using AlphaScreen assay to identify the host cell kinases that are cleaved by HIV-1 PR. We use two-color immunoblot to show the cleavage of TBK1 by HIV-1 extracellularly and used transfection studies and western blot to demonstrate that this also happens intracellularly. We identify the cleavage site by generating site specific substitution mutants and deletion mutants of TBK1.
We further use luciferase-based assays and immunofluorescence microscopy to demonstrate the effect of HIV-1 PR mediated TBK1 on downstream pathways involved in IFN-I production and the role of PIs in preventing this. Using drug resistant mutant HIV-1 PR we demonstrate that evolution of resistance to PIs can affect the ability of HIV-1 PR to cleave TBK1.
Results:
With AphaScreen assay we identify that TBK1 could be cleaved by HIV-1 PR. We further confirm that this cleavage not only happens extracellularly, but also intracellularly. Using sequencing and site-specific mutants, we show that HIV-1 PR cleaves TBK1 between the amino acids L683 and V684.
We demonstrate with the luciferase-based assay that HIV-1 PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity. We further show through immunofluorescence microscopy that in the presence of HIV-1 PR, TBK1 fails to cause the nuclear translocation of IRF3 which is the transcription factor responsible for IFN-I secretion.
We further reveal that the HIV-1 PR mediated suppression of IFN-I could be counteracted by PI in vitro. PIs can inhibit HIV-1 PR and increase IFN-I production as shown by the increase of IFN-I mRNA in PI treated productively infected cells compared to untreated cells. We also reveal that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme’s ability to cleave TBK1.
Discussion:
PR is an important enzyme of HIV-1 coded by the pol gene along with the other enzymes, reverse transcriptase, and integrase which help in viral replication (Konvalinka et al., 2015). However, there is accumulating evidence that HIV-1 PR plays an accessory role to interfere with host cellular functions by cleaving various intracellular proteins (Wagner et al., 2015). The salient observation in this regard is the proteolytic cleavage of host protein kinases involved in innate immunity by HIV-1 PR such as the nuclear Dbf2-related (NDR) kinases, NDR1 and NDR2 and the receptor interacting protein kinases (RIPK) RIPK1 (Devroe et al., 2005; Wagner et al., 2015). In similar lines, our study shows that TBK1 is yet another host PK that is cleaved by HIV-1 PR to inhibit IFN-I production possibly to dampen innate immunity and promote viral infection.
PIs are highly effective drugs against HIV-1 and comprise the mainstay agents of ART. With our in vitro experiments it is evident that PIs can prevent HIV-1 from cleaving TBK1, suggesting that PIs could improve the innate immune response to facilitate viral clearance. It has been observed that mutations of HIV-1 PR that enable the enzyme to become resistant to PIs paradoxically reduce the pathogenic fitness of the virus (de Vera et al., 2013). We were able to demonstrate that mutations in PR which confer resistance to PIs make the enzyme less potent to cleave TBK1 while still retaining its ability to cleave the viral polyprotein into functional units to cause viral maturation. We were able to demonstrate that mutations in PR which confer resistance to PIs make the enzyme less potent to cleave TBK1 while still retaining its ability to cleave the viral polyprotein into functional units to cause viral maturation.
The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.
