Abstract
The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.
Highlights
The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome AIDS and, over the course of the last century, has infected more than 70 million people worldwide
If the ubiquitin-like protein NEDD8 (Neural precursor cell expressed developmentally down-regulated protein 8) is attached to the Cullin-RING E3 ligase (CRL), it is activated, whereas removing NEDD8 leads to inactivation
The viral life cycle of HIV-1 is influenced by host proteins that either support or limit viral replication [2,5,6,7,8]
Summary
The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome AIDS and, over the course of the last century, has infected more than 70 million people worldwide. To undergo efficient replication in vivo, HIV-1, which only expresses a small set of viral proteins [1,2], has remarkably adapted to thrive in the hostile environment of the human host. Members of the cullin family serve as essential scaffolding proteins in the ubiquitin-proteasome system (UPS) and represent an important example of how HIV-1 manipulates host proteins to create a more amenable cellular environment. HIV-1 is known to use its accessory proteins Vpu, Vif and Vpr to hijack the function of several CRLs to counteract the human immune system and create a cellular environment which is beneficial for the virus [14]. HIV-2 and closely related simian immunodeficiency viruses (SIVs) hijack CRL4 with their accessory protein Vpx to ubiquitinate and degrade the antiviral protein SAMHD1 [22,23,24]
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