The immunosuppressive properties of the HIV Vpr protein are linked to a single highly conserved residue, R90

Abstract

Background: A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues. Consistent with this idea are reports that defects in the C-terminus of Vpr are associated with long-term non-progression.

Principal findings: Vpr RNA amplified from various sources was electroporated into monocyte-derived DC and IL-12 levels in supernatants were analyzed. The analysis of previously reported C-terminal Vpr mutations demonstrate that they do not alleviate the block of IL-12 secretion. However, a novel single conservative amino acid substitution, R90K, reverses the IL-12 suppression. Analysis of 1226 Vpr protein sequences demonstrated arginine (R) present at position 90 in 98.8%, with other substitutions at low frequency. Furthermore, none of sequences report lysine (K) in position 90. Vpr clones harboring the reported substitutions in position 90 were studied for their ability to suppress IL-12. Our data demonstrates that none of tested substitutions other than K relieve IL-12 suppression. This suggests a natural selection for sequences which suppress IL-12 secretion by DC and against mutations which relieve such suppression. Further analyses demonstrated that the R90K, as well as deletion of the C-terminus, directs the Vpr protein for rapid degradation.

Conclusion: This study supports Vpr as an HIV virulence factor during HIV infection and for the first time provides a link between evolutionary conservation of Vpr and its ability to suppress IL-12 secretion by DC. DC activated in the presence of Vpr would be defective in the production of IL-12, thus contributing to the prevailing Th2 cytokine profile associated with progressive HIV disease. These findings should be considered in the design of future immunotherapies that incorporate Vpr as an antigen.

Conflict of interest statement

Competing Interests: At the time of work conducted the following authors were employed by Argos: Irina Tcherepanova, Aijing Starr, Brad Lackford, Melissa D. Adams, David Calderhead, Don Healey and Charles Nicolette. Presently the following authors are employed by Argos: Irina Tcherepanova, David Calderhead, Don Healey and Charles Nicolette. The following authors own a stock options of Argos Therapeutics: Irina Tcherepanova, Aijing Starr, Brad Lackford, Melissa D. Adams, David Calderhead, Don Healey and Charles Nicolette. Argos Therapeutics Inc. is not publically traded Company. None of authors are members of the Board of Directors. The work is not associated with any pending or filed patent application. The funds did not come from Research grant, Travel grant or a Gift. The work was funded by Venture capital and the funders did not have any influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Figures

Figure 1. Alignment of amino acid sequence of Vpr amplified from isolate Vpr-2 and non infections plasmid pBKBH10S.

Alignment and alignment report were generated using Lasargene software (DNAstar). Amino acids 15, 37, and 55, outside of the C-terminal region which differ between the two Vpr proteins are highlighted by grey boxes. The last grey box (amino acid 74) indicates the divergence of the two sequences at the C-termini.

Figure 2. Full length but not truncated Vpr inhibits IL-12 secretion by DC.

Levels of IL-12 detected in supernatants of DC cultures 24 hrs post-electroporation. Black bar GFP, grey bar: 1 µg of RNA of Vpr truncated version pattern bar: 0.25 µg of full length Vpr (Vpr 2) were electroporated per million of DC. Condition when GFP RNA was electroporated alone is taken as maximum potential level of IL-12 cytokine secretion in each experiment.

Figure 3. Effect of mutants on IL-12 secretion and immunophenotype.

Analysis of IL-12 secretion profile and phenotype of the DC transfected with Vpr mutants in position 77 and LXXLL C-terminal motif. Full-length Vpr from either Vpr 2 or p98CN009.8 plasmid or its mutants, no Vpr with CD40L RNA (control) or GFP alone were electroporated into DC at amount of one microgram of RNA per million of DC. Panel A: levels of IL-12 cytokine detected in DC supernatants 24 hrs post- electroporation. Panel B: percent positive cells stained with specific anti CD80, anti CD83 and anti CD86 antibody. Panel C: mean fluorescent intensity of signal detected with specific antibody.

Figure 4. Vpr R90K mutation restores IL-12 secretion from DC.

Analysis of IL-12 secretion profile and phenotype of the DC transfected with Vpr mutants in C-terminal region. Full-length Vpr from p98CN009.8 plasmid or its mutants, or no Vpr with CD40L RNA (control) were electroporated into DC at amount of one µg of RNA per million of DC. Panel A: levels of IL-12 cytokine detected in DC supernatants 24 hrs post- electroporation. Panel B: mean fluorescent intensity of signal detected with specific antibody recognizing CD80, CD83, and CD86 surface markers. Viability of cells 24 hrs post electroporation with WT, Δ−C term, R90K ΔSRS or GFP RNAs were 93%, 93%, 94% 94% and 94% respectively.

Figure 5. Vpr mutants have enhanced ubiquitin-mediated degradation leading to low steady-state levels in DC.

Analysis of Vpr expression from RNA template in vitro and in DC. Panel A:Western blot analysis of in vitro-translated full-length Vpr RNA or its mutations as indicated. NTC: control reaction performed in the absence of an RNA template. Panel B: Western blot analysis on lysates of DC electroporated with full length Vpr RNA or its mutations. After electroporation DC were incubated for four hours in the absence or presence of lactacystin at 10 µM final concentration as indicated. IVT VPR Ctrl: in vitro-translated full-length Vpr protein used as a positive control in Western blot assay. RNAs used for electroporation of DC are indicated on the top.

Figure 6. Analysis of amino acid substitutions reported in Los Alamos database on Vpr ability to modulate IL-12.

Vpr clones carrying the most frequently reported amino acid substitutions in position 90 of 1,226 full-length Vpr sequences in the Los Alamos National Database were cloned and tested for their ability to modulate IL-12 secretion by the DC. Vpr wild-type (WT), VPR lacking C-terminus (ΔCterm), Vpr R90K, Vpr R90G, Vpr R90S RNAs or GFP RNA alone were electroporated into DC and levels of IL-12 detected in DC supernatants 24 hrs post-electroporation. Viability of cells electroporated with WT and mutant Vpr RNA was 71–85% whereas the viability of a GFP RNA-electroporated DC was 76%.