Characteristics of HIV-1 envelope mutations after IFN-γ treatment <i>in</i><i>vivo</i>.

<p>(<b>A</b>) Frequency of modification occurring at adenosines with different 5′ nearest neighbors. Adenosine in the preferential selection of 5′ neighbors for ADAR1 (A>>U = G>C). (<b>B</b>) Numbers of amino acid substitutions around HIV-1 envelope glycoprotein V3 region. X-axis number is the amino acid position is counted from N-terminus of the envelope protein. (<b>C</b>) Amino acid substitutions resulted from by A to G mutations are shown in red.</p

Comparison of total positive charges in the V3 loops of viruses isolated from patients at pre- and post- IFN-γ treatment.

<p>No increase in positive charges was observed in any patients after the treatment.</p

A diagram showing the distinct targets of adenosine and cytidine deaminase in the HIV-1 life cycle.

<p>IFN-γ induces both adenosine deaminase (ADAR1L) and cytidine deaminase (APOBEC3G). Adenosine deaminase may inhibit the step after viral transcription whereas cytidine deaminase acts on reverse transcription immediately after virus entry.</p

Reduction of lung HIV-1 RNA levels and HIV-1 envelope mutations associated with aerosol IFN-γ treatment.

<p>(<b>A</b>) HIV-1 RNA levels in bronchoalveolar lavage (BAL) fluids of 5 HIV-1/<i>M. tb</i> co-infected patients before (pre IFN-γ) and after (post IFN-γ) treatment with aerosolized IFN-γ. HIV-1 RNA viral load at post-treatment was significantly reduced in BAL fluids (p<0.05; mean±SE). (<b>B</b>) Nucleotide mutations around the V3 region of HIV-1 envelope post IFN-γ treatment. The percentages of mutations found in the V3 region were calculated by comparing virus sequences before and after IFN-γ therapy. A to G and G to A mutation occurred significantly as compared with other mutations (p<0.01).</p

ADAR1 overexpression and knock down in a chronically HIV-1 infected macrophage cell model system.

<p>(<b>A</b>). Chronic HIV-1 infection induced ADAR1 <i>in</i><i>vitro</i>. Expression of 150 kD and 110 kD ADAR1 was measured in OM10.1 cells and YS+OA cells treated with and without indinavir (IDV). OM10.1 is a latently HIV-1 infected macrophage cell line (HL-60 cell is the parental uninfected cell line). YS+OA cells are OM10.1 cells that stably over-express ADAR1. YS+OA cells have increased expression of the ADAR1 150 kDa isoform. Densitometry data are shown below the lanes. (<b>B</b>) HIV-1 RNA copies in the culture supernatant from ADAR1-overexpressing YS+OA were significantly lower than those of ADAR1-knocked down YS-OA. (p<0.05; mean±SE, n = 6). YS-OA is OM10.1 cells that are stably transfected with shRNA to knock down of ADAR1. (<b>C</b>) Intra-cellular p24 gag and its precursor Pr55 protein in ADAR1-overexpressing YS+OA as compared to ADAR1-knocked down YS-OA and untreated OM10.1. (<b>D</b>) Nuclear run-on transcription assays of YS+OA cells and YS-OA cells. Synthesis of HIV-1 nascent RNA was calculated as the ratio of HIV-1 RNA to GAPDH RNA.</p

ADAR1 expression in primary macrophages and T cells <i>in</i><i>vitro</i>.

<p>(<b>A</b>) IFN-γ induced 150-kDa ADAR1L in MDM. Cells were treated for six hours with the indicated concentrations of IFN-γ or infected with HIV-1 overnight. Immune-blot of actin was used as loading control. The ratio 150-kDa (IFN inducible) ADAR1L isoform to 110-kDa (constitutive) ADAR1S is shown below each lane. (<b>B</b>) IFN-γ did not induce ADAR1L in primary CD4+ T cells. CD4+ T cells were incubated with IFN-γ or HIV-1 as in panel (<b>A</b>).</p

Comparison of mutations around the V3 region of HIV envelope in viruses isolated <i>in</i><i>vivo</i> (Fig. 1B) versus mutations in viruses produced <i>in</i><i>vitro</i> from the ADAR1-overexpressing chronically HIV-infected YS+OA cell line.

<p>Comparison of mutations around the V3 region of HIV envelope in viruses isolated <i>in</i><i>vivo</i> (Fig. 1B) versus mutations in viruses produced <i>in</i><i>vitro</i> from the ADAR1-overexpressing chronically HIV-infected YS+OA cell line.</p

ADAR1 mRNA expression in the human lung.

<p>(<b>A</b>) ADAR1 mRNA levels were higher in HIV-1-infected patients before anti-retroviral treatment and reduced to normal after treatment (p<0.01, mean±SE). ADAR1 mRNA expression was measured by high-density cDNA arrays. Before anti-retroviral therapy and after 4 weeks of antiretroviral therapy in BAL cells of the same patients. Normal indicates mRNA from BAL cells of uninfected volunteers. (<b>B</b>) ADAR1 mRNA expression was measured by high-density cDNA arrays in 8 patients before and one-month after treatments with aerosolized IFN-γ. IFN-γ treatment was associated with about 2 fold increase in of ADAR1 mRNA (0.15±0.03 vs 0.25±0.07; mean±SE).</p

HIV-1 replication following ADAR1 siRNA knockdown in MDM infected <i>in</i><i>vitro</i> and alveolar macrophages from HIV-1 seropositive patients.

<p>(<b>A</b>) Knockdown of ADAR1 (L+S) by siRNA in MDM. siRNA for ADAR1 led to attenuated expression of both isoforms while siRNA specific to ADAR2 did not attenuate expression of either isoform as compared to negative control. (<b>B</b>) Induction of HIV-1 replication in MDM after ADAR1 siRNA treatment. 50% tissue culture infectious dose (TCID₅₀) infectivity assay was used to measure virus infectivity in the supernatant of siRNA treated MDM. TCID₅₀ assay was performed 5 days after HIV-1 infection. Infectivity was normalized to that of control. No induction of virus replication was seen in cells treated with ADAR2 siRNA. (<b>C</b>) Induction of HIV-1 replication by ADAR1 siRNA in alveolar macrophages of four HIV-infected patients on antiretroviral therapy. Two days after the knock down of ADAR1 gene with siRNA, the amount of virus in the culture supernatant was significantly increased as compared to ADAR2 siRNA control (p<0.05).</p