Abstract
BackgroundHIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modulation of several host cell functions. In addition to pro-apoptotic and cytostatic properties, Vpr can redirect cellular E3 ubiquitin ligases (such as DCAF1-Cul4A E3 ligase complex) to target many host proteins and interfere with their functions. Among them, Vpr binds the uracil DNA glycosylase UNG2, which controls genome uracilation, and induces its specific degradation leading to loss of uracil removal activity in infected cells. Considering the essential role of UNG2 in antibody diversification in B-cells, we evaluated the impact of Vpr on UNG2 fate in B lymphocytes and examined the functional consequences of UNG2 modulations on class switch recombination (CSR).MethodsThe impact of Vpr-induced UNG2 deregulation on CSR proficiency was evaluated by using virus-like particles able to deliver Vpr protein to target cells including the murine model CSR B cell line CH12F3 and mouse primary B-cells. Co-culture experiments were used to re-examine the ability of Vpr to be released by HIV-1 infected cells and to effectively accumulate in bystander B-cells. Vpr-mediated UNG2 modulations were monitored by following UNG2 protein abundance and uracil removal enzymatic activity.ResultsIn this study we report the ability of Vpr to reduce immunoglobulin class switch recombination (CSR) in immortalized and primary mouse B-cells through the degradation of UNG2. We also emphasize that Vpr is released by producing cells and penetrates bystander B lymphocytes.ConclusionsThis work therefore opens up new perspectives to study alterations of the B-cell response by using Vpr as a specific CSR blocking tool. Moreover, our results raise the question of whether extracellular HIV-1 Vpr detected in some patients may manipulate the antibody diversification process that engineers an adapted response against pathogenic intruders and thereby contribute to the intrinsic B-cell humoral defect reported in infected patients.
Highlights
HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modula‐ tion of several host cell functions
We report the capacity of Vpr to decrease class switch recombination (CSR) capacities of stimulated mouse CH12F3 cells, a result that was further validated in primary B-cells
Anti-hUNG2 rabbit polyclonal NBP1-49985 was from Novus Biologicals; anti-Vpr mixture was made with equal amounts of goat polyclonal antibody from Santa Cruz Biotechnology, rabbit polyclonal antibody #709 and #11836; anti-α tubulin mouse monoclonal antibody (T5168) was from Sigma; goat polyclonal anti-p24 (49999007) was from AbD Serotec; Secondary HRP-conjugated antibodies were from Jackson Laboratory
Summary
HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modula‐ tion of several host cell functions. Vpr induces cell cycle arrest of infected cells at the G2/M phase [11] This last extensively described function [12], has been initially associated with the capacity of Vpr to activate the ataxia telangiectasia-mutated (ATM) [13] and Rad3-related proteins (ATR) and to stimulate phosphorylation of ATR substrates Chk and H2AX histone variants [14]. Recently, this cytostatic property has been correlated with the activation of the structurespecific endonuclease regulator SLX4 complex by Vpr [15]. Vpr induces apoptosis through diverse mechanisms, either related to mitochondrial dysfunction, or by interacting with Bax or the voltage-dependent anion channel (VDAC), all leading to cytochrome c release and caspase 3 activation [16]
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