HIV-1 and <i>Mycobacterium bovis</i>BCG infections increased the transcript levels of hZNF-134.

<p>PBMCs were isolated from three healthy donors and infected either with HIV, <i>Mycobacterium bovis</i>BCG or both. The levels of hZNF-134 was measured by qRT-PCR and compared to uninfected controls. (A) hZNF-134 transcript levels upon HIV infection. (B) hZNF-134 transcript levels upon <i>Mycobacterium bovis</i>BCG infection. (C) Comparison of hZNF-134 transcript levels upon HIV and HIV-<i>Mycobacterium bovis</i>BCG co-infections. Uninfected cells were used as controls. The transcript levels were normalized to transcript levels of β-Actin. (D) The relative expression of 9 kb viral transcript was measured in HIV and HIV-<i>Mycobacterium bovis</i>BCG co-infections. The transcript levels were normalized to transcript levels of β-Actin. All experiments were done in triplicate and error bars represent mean ± SD.</p

hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells.

<p>HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.</p

hZNF-134 could bind to HIV-1 LTR.

<p>(A) GFP or hZNF-134-GFP fusion proteins were transiently expressed in HEK293T cells. After 48 hours post transfection, cell lysates were prepared and Western blot with anti-GFP antibody was performed to check for the expression of GFP or hZNF-134-GFP. GAPDH was used as a loading control. (B) Transiently expressed GFP or hZNF-134-GFP in HEK293T cells were used for pull-down assays with biotinylated LTR. Western blot analyses of the pull-down samples using anti-GFP antibody showed that biotinylated LTR could capture hZNF-134-GFP successfully but GFP alone was not captured by LTR. Pull-down experiments were performed more than three times.</p

Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

<div><p>Background</p><p>Concurrent occurrence of HIV and Tuberculosis (TB) infections influence the cellular environment of the host for synergistic existence. An elementary approach to understand such coalition at the molecular level is to understand the interactions of the host and the viral factors that subsequently effect viral replication. Long terminal repeats (LTR) of HIV genome serve as a template for binding trans-acting viral and cellular factors that regulate its transcriptional activity, thereby, deciding the fate of HIV pathogenesis, making it an ideal system to explore the interplay between HIV and the host.</p><p>Methodology/Principal Findings</p><p>In this study, using biotinylated full length HIV-1 LTR sequence as bait followed by MALDI analyses, we identified and further characterized human-Zinc-finger-protein-134 (hZNF-134) as a novel positive regulator of HIV-1 that promoted LTR-driven transcription and viral production. Over-expression of hZNF-134 promoted LTR driven luciferase activity and viral transcripts, resulting in increased virus production while siRNA mediated knockdown reduced both the viral transcripts and the viral titers, establishing hZNF-134 as a positive effector of HIV-1. HIV, <i>Mycobacteria</i> and HIV-TB co-infections increased hZNF-134 expressions in PBMCs, the impact being highest by mycobacteria. Corroborating these observations, primary TB patients (n = 22) recorded extraordinarily high transcript levels of hZNF-134 as compared to healthy controls (n = 16).</p><p>Conclusions/Significance</p><p>With these observations, it was concluded that hZNF-134, which promoted HIV-1 LTR activity acted as a positive regulator of HIV propagation in human host. High titers of hZNF-134 transcripts in TB patients suggest that up-regulation of such positive effectors of HIV-1 upon mycobacterial infection can be yet another mechanism by which mycobacteria assists HIV-1 propagation during HIV-TB co-infections. hZNF-134, an uncharacterized host protein, thus assumes a novel regulatory role during HIV-host interactions. Our study provides new insights into the emerging role of zinc finger proteins in HIV-1 pathogenesis.</p></div

hZNF-134 over expression enhanced the transcriptional potential of LTR and increased viral titers.

<p>(A) Luciferase activity was measured in the presence or absence of Tat following transfection with pEGFP-C3 or ZNF-134-GFP-C3 vector into HEK293T cells along with the reporter construct, pLTR-luc. Luciferase activity was measured in terms of relative luminescence units (RLU) from HEK293T cell lysates after 48 hours and plotted as fold increase in the luciferase activity. Cells over-expressing hZNF-134-GFP showed increased luciferase activity when compared to GFP alone. (B) pNL4-3 was transfected in HEK293T cells with either GFP or hZNF-134-GFP. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts increased in the presence of over-expressed hZNF-134. (C) HEK293T cells over-expressing either GFP or hZNF-134-GFP were transfected with pNL4-3 vector and after 48 hours scored for viral p24 by ELISA. Inset shows the Western blot confirming the over-expression of either GFP or hZNF-134-GFP in HEK293T cells. For loading control, expression of GAPDH was detected by Western blots in both the cases. Over-expression of hZNF-134 increased viral titers. All the transfection efficiencies were normalized to pRL-TK transfections. All experiments were done more than three times and error bars represents mean ± SD. Student's <i>t</i>-test was performed and *p<0.05 was considered significant.</p

Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-α and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

<div><p>Background</p><p>Cytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy.</p><p>Methodology/Principal Findings</p><p>We analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n = 57), TB infected (n = 57), HIV infected (n = 59) and HIV-TB co-infected (n = 57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and <i>vice-versa</i>. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio.</p><p>Conclusion/Significance</p><p>With these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.</p></div

siRNA mediated knockdown of hZNF-134 decreased the viral transcript levels and the final viral titer output measured by p24 ELISA.

<p>(A) qRT-PCR showing reduced transcript levels of hZNF-134 after siRNA mediated knockdown. Scrambled siRNA was used as a control. 50picomoles of each siRNA were used. The transcript levels were normalized to the β-actin levels. (B) pNL4-3 was transfected in HEK293T cells treated with 50 picomoles of scrambled siRNA or hZNF-134 siRNA. After 48 hours, the viral transcription was measured by qRT-PCR. The plot shows that viral transcripts decreased following the knockdown of hZNF-134. (C) Viral p24 levels were measured in pNL4-3 transfected HEK293T cells after treatment with 50picomoles of hZNF-134 specific or scrambled siRNA. All experiments were done more than three times and error bars represents mean ± SD. Student's <i>t</i>-test was performed and *p<0.05 was considered significant.</p

Comparison of anti-inflammatory cytokines Box plots representing plasma levels of (A) IL-4, (B) IL-10 (C) Ratio of IFN-γ/IL-10 in Healthy, TB, HIV and HIV-TB categories.

<p>The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.</p

Blood plasma levels of cytokines Box plots representing blood plasma levels of (A) IL-12 p70, (B) IL-2 in the Healthy, TB, HIV and HIV-TB categories.

<p>The threshold for significance was set at p≤0.05. Bars above and below the plots represent the statistical significance (p value) between the groups.</p

Correlation of CD4+T cells and viral load as indicators of HIV progression.

<p>Box plots representing (A) the distribution of CD4+T cell/mm³ of blood in Healthy, HIV and HIV-TB. (B) The distribution of viral RNA copies measured in terms of IU/ml of sample for the HIV and HIV-TB patients. (C) Analyses of the CD4+T cell count of HIV and HIV-TB samples with low viral load (VL≤10⁵ IU/ml) and high viral load (VL>10⁵IU/ml). The threshold for significance was set at p≤0.05. Bars above the plots represent the statistical significance (p value) between the groups.</p