The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G

Abstract Background The HIV-1 accessory protein known as viral infectivity factor or Vif binds to the host defence factor human APOBEC3G (hA3G) and prevents its assembly with viral particles and mediates its elimination through ubiquitination and degradation by the proteosomal pathway. In the absence of Vif, hA3G becomes incorporated within viral particles. During the post entry phase of infection, hA3G attenuates viral replication by binding to the viral RNA genome and deaminating deoxycytidines to form deoxyuridines within single stranded DNA regions of the replicated viral genome. Vif dimerization has been reported to be essential for viral infectivity but the mechanistic requirement for Vif multimerization is unknown. Results We demonstrate that a peptide antagonist of Vif dimerization fused to the cell transduction domain of HIV TAT suppresses live HIV-1 infectivity. We show rapid cellular uptake of the peptide and cytoplasmic distribution. Robust suppression of viral infectivity was dependent on the expression of Vif and hA3G. Disruption of Vif multimerization resulted in the production of virions with markedly increased hA3G content and reduced infectivity. Conclusion The role of Vif multimerization in viral infectivity of nonpermissive cells has been validated with an antagonist of Vif dimerization. An important part of the mechanism for this antiretroviral effect is that blocking Vif dimerization enables hA3G incorporation within virions. We propose that Vif multimers are required to interact with hA3G to exclude it from viral particles during their assembly. Blocking Vif dimerization is an effective means of sustaining hA3G antiretroviral activity in HIV-1 infected cells. Vif dimerization is therefore a validated target for therapeutic HIV-1/AIDS drug development.</p

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-0

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>ither AZT (1 μM), Control peptide (50 μM), Peptide 1 (50 μM) or Peptide 2 (50 μM) or left untreated (viral control) as described in Methods. At the indicated days post-infection, cells were harvested for cell lysate preparation and reverse transcriptase quantification as described in Methods. Lysates were prepared from parallel cultures of uninfected and untreated cells (cell control) as controls for the reverse transcriptase assays

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-4

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>0 μM Peptide 2 for 24 hours and processed for FACS analysis as described in Methods. The percent of cells in G1, S and G2/M phases of the cell cycle were calculated based on the DNA staining distributions

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-3

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p> with or without A3G (as indicated below each histogram). During the incubation period, Peptide 2 was dosed into the specified samples (50 μM final concentration). The viral particles collected from the cell culture media over 48 h were normalized for their p24 content and incubated with JC53-bl cells for analysis of infectivity corresponding to luminescence as described in Methods. Infectivity of +Vif virions is shown as percent of the infectivity measured for +Vif/-hA3G/-peptide condition (14,498 red units). The (-) A3G/ΔVif virus control virions lacked Vif and the cells did not express A3G. Infectivity of the ΔVif virions is shown as percent of the infectivity measured with ΔVif/-hA3G/-peptide conditions (5,827 red units). The error bars represent the standard deviation with n = 3

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-5

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>ith and without Peptide 2 treatments (50 μM), were normalized for p24 and sedimented through a sucrose cushion as described in Methods. The resultant pellets were lysed and resolved via SDS-PAGE and western blotted for A3G and p24. P24 was re-probed in the western blot in addition to the p24 ELISA quantification to validate the normalization. () Virion samples harvested from the co-transfection were normalized for p24 and infected into JC53-bl cells to quantify infectivity by luminescence analysis as described in Methods. Bars represent standard deviations with an n = 3

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-2

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>unted with DAPI-containing media and fluorescence microscopy was performed with filters for DAPI and FITC as described in Methods. Longer durations of treatment were also evaluated in a similar manner. The images were manually overlaid to superimpose the image of the nucleus with each image of Peptide 2-FITC distribution in the cell. Cells from two different regions of the H9 and MT2 plates are shown

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-7

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>ty assayed as described in Methods. The percent inhibition of viral infectivity by the peptide was determined during the first seven days of the spreading infection assay. The Inhibitor Concentration, IC (indicated within each histogram) was calculated relative to the untreated virus control

The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G-1

<p><b>Copyright information:</b></p><p>Taken from "The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G"</p><p>http://www.retrovirology.com/content/4/1/81</p><p>Retrovirology 2007;4():81-81.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2222665.</p><p></p>ty assayed as described in Methods. The percent inhibition of viral infectivity by the peptide was determined during the first seven days of the spreading infection assay. The Inhibitor Concentration, IC (indicated within each histogram) was calculated relative to the untreated virus control

Dual mRNA therapy restores metabolic function in long-term studies in mice with propionic acidemia

Propionic acidemia is a serious pediatric inherited disorder with no effective treatments. Here the authors demonstrate that delivering dual mRNAs as an enzyme replacement approach can be used as an effective therapy in a mouse model of propionic acidemia, with potential applicability to chronically administer multiple mRNAs in other genetic disorders