HIV-1 Vpr suppresses the cytomegalovirus promoter in a CRL4(DCAF1) E3 ligase independent manner

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Highlights

  • HIV-1 Vpr is a transcriptional suppressor of the CMV promoter.

  • CRL4(DCAF1) E3 ligase is not essential for Vpr inhibition of the CMV promoter.

  • Sites A30/V31 of HIV-1 Vpr are essential for suppression of the CMV promoter.

  • Sites A30/V31 of HIV-1 Vpr are required for transcriptional regulation of UNG2.

Abstract

Although the Vpr protein of human immunodeficiency virus type 1 (HIV-1) has been shown to act as a transcriptional activator of the HIV-1 LTR and certain host genes, the current study demonstrates that it can also function as a potent inhibitor of the cytomegalovirus (CMV) promoter. Previous studies have shown that the cell cycle arrest and apoptotic functions of Vpr required recruitment of the CRL4(DCAF1) E3 ligase, but this complex is shown not to be required for inhibition of the CMV promoter. We identified conserved sites (A30/V31) from diverse Vpr from HIV/SIV that were critical for blocking the CMV promoter activity. Interestingly, the Vpr mutant A30S/V31S protein also impaired the ability of Vpr to down-regulate transcription of the host UNG2 gene. Our findings shed light on the dual functions of Vpr on the transcription of HIV-1, other viruses and host genes which may contribute to viral replication and disease progression in vivo.

Introduction

Human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV) encode a conserved 14 KDa protein of 96 amino acids, the regulatory viral protein R (Vpr) [1], [2], [3], [4]. Vpr and its homologous viral protein Vpx which is encoded by HIV-2 and SIV, are specifically packaged into virus particles through an interaction with the p6 region of the Gag precursor, indicating a role in the early steps of the virus life cycle [1], [3], [5], [6]. More importantly, in SIV-infected monkeys, Vpr is required for viral dissemination and disease progression [7], [8], [9].

Vpr is known to be a multifunctional viral protein. The most investigated function of Vpr is its ability to prevent cell proliferation and arrest the cell cycle at the G2 phase [10]. Other HIV-1 accessory proteins, such as Vif and Vpu, have been shown to function by hijacking of the cellular E3 ubiquitin ligase to degrade the host restriction factors, APOBEC3G and BST-2/tetherin, respectively. By a similar mechanism Vpr has been shown to function by recruiting the host CRL4(DCAF1) E3 ligase to induce cell cycle arrest by degrading an as yet unidentified factor [11], [12], [13], [14], [15]. Recent studies have also indicated that Vpr interacts with and prematurely activates the host SLX4/MUS81/EME1 complex, which promotes cell cycle arrest and evasion of the innate immune response [16], [17].

Vpr also triggers apoptosis of host cells, which contributes to the depletion of CD4+ T cells in vivo [18], [19], [20]. Although Vpr is not required for infection of activated T cells, it enhances viral replication in non-dividing monocytes [21], [22]. In addition, Vpr also has several other functions such as dysregulation of mitochondria [23], [24] and nuclear import of the viral preintergration complex [25].

Increasing evidence suggests that Vpr is a transcriptional regulator of the HIV-1 LTR [26] and an array of host genes, including ung2 [27], nlbp2 [28], nhe1 [29] among others, which may enhance viral replication and disease progression in vivo. In the current study, we show that Vpr is a transcriptional suppressor of the promoter of cytomegalovirus (CMV), which is widely distributed in latent form among human populations. Further studies indicate that conserved sites in the first helix of HIV-1 Vpr are essential for Vpr-mediated inhibition of the transcription of both CMV and the host gene ung2. The potential implications for HIV-1 disease progression of Vpr's effect on viral and host gene transcription are discussed.

Section snippets

Plasmid construction

The sequence of Vpr from HIV-1 NL4-3 stain cloned into the expression vector VR1012 with an HA tag at the N-terminus has been described previously [30].pVpr A30S/V31S and Q65R were made from pVpx-HFA by site-directed mutagenesis. Plasmids pSIVmus Vpr and pSIVdeb Vpr established in plasmid VR1012 has been described previously [31]. pCMV-GFP, with GFP sequences in the pcDNA3.1 vector, was purchased from Invitrogen (V795-20). The ung2 promoter reporter vector pung2-Luc S was a gift from Geir

Vpr inhibits the transcription activity of the CMV promoter

CMV, a herpes virus, is highly prevalent in human populations as an asymptomatic infection. However, in immunocompromised individuals, such as those with AIDS, it is a major cause of morbidity and mortality [32]. Vpr has been shown to transactivate HIV-1 transcription and the affinity of its binding to the HIV-1 LTR has been correlated with specific virulence properties of the virus [33], [34], [35], [36]. Given the adverse effects of CMV infection in the setting of immunodeficiency induced by

Conflict of interest

None.

Acknowledgments

We thank Dr. Geir Slupphaug for critical reagents, Zhaolong Li, Chunyan Dai for technical assistance. This work was supported in part by funding from the Chinese Ministry of Science and Technology (No 2012CB911100) and the Chinese Ministry of Education (No IRT1016) and from Tianjin University.

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  • 1

    These authors contributed equally to this work.

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