Virion-packaged A3G and infectivity of endogenous viruses produced from activated CD4+ T lymphocytes <i>ex vivo</i>.

<p>(<b>A</b>) Increased virion-packaged A3G protein was associated with decreased infectivity of endogenous viruses produced from activated CD4+ T lymphocytes. Virions released from activated CD4+ T lymphocytes of ART-suppressed (AS) non-controller subjects were analyzed for infectivity using the TZM-bl assay and for virion-associated A3G protein by in-virion Western blotting. CD4+ T lymphocytes were sorted for positivity for CD25, CD69, CD38 and HLA-DR from blood of 5 of the AS non-controller subjects. Activation was maintained in <i>ex vivo</i> cultures by anti-CD3/CD28 antibody coated beads. HIV-1 p24 antigen measurement monitored production of endogenous Vif-positive HIV-1 <i>ex vivo</i>. Virion content was normalized by p24 antigen amount for both infectivity and virion A3G protein measurements. Bars represent mean +/− standard deviation (SD). (<b>B</b>) Virion infectivity was significantly inversely correlated with amount of A3G protein in virions. P value was determined by Spearman correlation.</p

Lower HIV Provirus Levels Are Associated with More APOBEC3G Protein in Blood Resting Memory CD4+ T Lymphocytes of Controllers <i>In Vivo</i>

<div><p>Immunodeficiency does not progress for prolonged periods in some HLA B57- and/or B27-positive subjects with human immunodeficiency virus type 1 (HIV) infection, even in the absence of antiretroviral therapy (ART). These “controllers” have fewer HIV provirus-containing peripheral blood mononuclear cells than “non-controller” subjects, but lymphocytes that harbor latent proviruses were not specifically examined in studies to date. Provirus levels in resting memory cells that can serve as latent reservoirs of HIV in blood were compared here between controllers and ART-suppressed non-controllers. APOBEC3G (A3G), a cellular factor that blocks provirus formation at multiple steps if not antagonized by HIV virion infectivity factor (Vif), was also studied. HLA-linked HIV control was associated with less provirus and more A3G protein in resting CD4+ T central memory (Tcm) and effector memory (Tem) lymphocytes (provirus: p = 0.01 for Tcm and p = 0.02 for Tem; A3G: p = 0.02 for Tcm and p = 0.02 for Tem). Resting memory T cells with the highest A3G protein levels (>0.5 RLU per unit of actin) had the lowest levels of provirus (<1,000 copies of DNA per million cells) <i>in vivo</i> (p = 0.03, Fisher's exact test). Using two different experimental approaches, Vif-positive viruses with more A3G were found to have decreased virion infectivity <i>ex vivo</i>. These results raise the hypothesis that HIV control is associated with increased cellular A3G that may be packaged into Vif-positive virions to add that mode of inhibition of provirus formation to previously described adaptive immune mechanisms for HIV control.</p></div

Higher cellular A3G protein levels were associated with decreased infectivity and spread of HIV virions after <i>ex vivo</i> activation and infection of primary memory T lymphocytes.

<p>(<b>A</b>) Blood resting CD4+ T central memory (Tcm) and effector memory (Tem) cells sorted from the same uninfected subject were activated and then infected <i>ex vivo</i> with a Vif-positive, CCR5-tropic clinical isolate of HIV. HIV p24 antigen was monitored over time in supernatant fluids from separate Tcm and Tem cell cultures. Virus spread through the culture of Tcm cells more rapidly than it did through the culture of Tem cells previously shown to have higher A3G protein levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076002#pone-0076002-g002" target="_blank">Figure 2</a>). (<b>B</b>) Infectivity of virions produced from Tcm cells was greater than that of virions produced from Tem cells. Infectivity was quantified by luciferase activity normalized by amount of p24 antigen added to TZM-bl reporter cells. Bars represent mean +/− standard deviation (SD).</p

HIV provirus (A) and APOBEC3G protein (B) levels in blood resting CD4+ T memory lymphocytes.

<p>(<b>A</b>) Blood resting CD4+ T central memory (Tcm) and effector memory (Tem) cells from viremic controller (VC) subjects (n = 7) each had lower HIV provirus copy numbers than those cells from antiretroviral therapy suppressed (AS) non-controller subjects (n = 6). <i>Alu</i>-PCR was used to determine HIV provirus copy numbers, as log integrated copies per million cells. (<b>B</b>) Blood resting CD4+ Tcm and Tem cells from VC subjects (n = 4) each had higher APOBEC3G (A3G) protein levels than those cells from AS non-controller subjects (n = 4). Relative light units (RLU) of A3G bands on immunoblots were quantified using Licor Odyssey, with normalization to β-actin. Cells from which A3G protein levels were determined were from 8 of the same time points studied for each subject in Fig. 1(A) (4 for VC and 4 for AS subjects); protein quantitation was possible for only a subset of the subject cells/time points studied in Fig. 1(A). In (<b>A</b>) and (<b>B</b>), lines indicate median values. P values were determined by 2-tailed Mann-Whitney test.</p