Inhibition of infection of incoming HIV-1 virus by RNA-cleaving DNA enzyme

AbstractNine different DNA enzymes (DzV3-n, n=1–9) targeting the V3 loop region of HIV-1 HXB2 were synthesized. One of those, DzV3-9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro. DzV3-9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co-receptors. The inhibitory effect of DNAzyme on incoming HIV-1 was also demonstrated with pseudotype virions generated by NL432-based luciferase reporter genes. Thus, an efficient, stable DNAzyme against a functionally important region of HIV-1 was identified, and it may be useful for prevention of HIV-1 infection

Blood monocytes harbor HIV type 1 strains with diversified phenotypes including macrophage-specific CCR5 virus

BACKGROUND: Recent studies have shown that blood monocytes harbor human immunodeficiency virus type 1 (HIV-1) variants that are genotypically distinguishable from those in CD4(+) T cells. However, the biological function of monocyte-derived HIV-1 remains unclear. METHODS: Using pseudovirus assay, we analyzed the phenotype conferred by monocyte-derived HIV-1 envelopes from 8 patients. RESULTS: All pseudoviruses carrying monocyte-derived HIV-1 envelopes used CCR5; however, their use of additional coreceptors delineated 4 phenotypes in which viruses used (1) CCR5 only, (2) CCR5 and CXCR4, (3) CCR3 and CCR5, or (4) multiple coreceptors, including CCR1, CCR3, GPR15, CCR5, and CXCR4. More importantly, we observed 2 distinct cell tropism phenotypes for pseudoviruses carrying monocyte-derived envelopes: (1) monocyte-derived, macrophage-specific R5 (MDMS-R5) virus that, using CCR5 only, could infect monocyte-derived macrophages (MDMs) but not CD4(+) T cells and (2) dual tropic virus that infected both MDMs and primary CD4(+) T cells. We found blood monocytes harboring viruses with multiple phenotypes as early as 25 days before seroconversion and as late as 9 years after seroconversion. CONCLUSIONS: These data suggest that HIV-1 circulating in blood monocytes represents diverse HIV-1 with multiple phenotypes and that MDMS-R5 viruses may play an important role in infection with and persistence of HIV-1 within the monocyte/macrophage lineag

Successful isolation of infectious and high titer human monocyte-derived HIV-1 from two subjects with discontinued therapy.

HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as "Trojan horse" hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes.In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays.These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages

Viral isolation from Subject 1175 and 1690.

<p>Clinical information of study Subject 1175 (A) and 1690 (B). For each subject, the first visit with confirmative diagnosis of seroconversion, along with the corresponding HIV-1 RNA viral load, is plotted at Month 0. Subsequently, longitudinal visits, viral loads, treatment and sampling time points are shown. (C) The monocyte purification strategy. Non-monocytes were magnetically labeled and removed from PBMCs of 2 healthy donors. The eluent monocytes were positively sorted and subjected to a four-hour attachment purification followed by two intensive washes with PBS. Percentage of T cell contamination before and after attachment purification was shown in the shaded quadrants of each flow cytometry graph. Monocyte- and CD4+ T cell-derived HIV-1 were obtained from subject 1175 (D) and 1690 (E) by being co-cultivated with CD8+ T cell-depleted donor PBMC. Longitudinal HIV-1 p24 concentrations in supernatants were shown for each co-cultivation. The high limit cut-off of HIV-1 p24 concentration is 20 ng/ml and lower limit for detection (LLD) is 0.01 ng/ml. The time points for viral expansion and stock preparation were indicated with dash lines with arrowheads. TCID₅₀ titers of each viral expansion of 1175 T and 1175 M (D), as well as 1690 T and 1690 M (E), are shown in each corresponding box.</p

Observation of replication kinetics in MDM.

<p>HIV-1 p24 production kinetics in MDM was shown for1690 M (A) and1690 T and 1175 M and 1175 T (B). Seven-day old MDM infection at MOI = 0.01 for 4 h before continuously cultured in fresh media. Supernatant HIV-1 p24 concentrations were determined and shown longitudinally (ng/mL, mean ± SEM, donor n = 4). (C) Supernatant viral RNA copy numbers of MDM infections. Supernatant samples were acquired on Day 22 post-infection and data were shown as copies/mL (donor n = 3). (D) Viral replication in PHA-PBMC. Donor PBMCs were stimulated by PHA for 48 h before BaL, 1690 M, 1690 T, 1175 M and 1175 T infections at MOI = 0.01. HIV-1 p24 concentrations in culture supernatants on Day 7 post-infection were shown (ng/mL, mean ± SEM). *<i>P</i><0.05, donor n = 4 compared with the BaL-infected group. <i>E,</i> Intracellular staining of HIV-1 p24. MDM were infected for 14 d before cyto-immunochemistry staining with CD14 (red), DAPI (blue) and HIV-1 p24 (green). Scale bar = 20 µm.</p

Intracellular viral DNA persistence in MDM acute infection and longitudinal reverse transcriptase activity assay.

<p>(A) HIV-1 DNA synthesis kinetics in MDM. Seven-day old MDM from 3 healthy donors were infected by 1690 M and 1690 T, respectively, at MOI = 0.01. Cells were harvested at 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 168 h post-infection. Intracellular HIV-1 DNA copy numbers were determined. *<i>P</i><0.05, donor n = 3. (B) Viral resistance to azidothymidine. Seven-day old MDM were infected by 1690 M and 1690 T, respectively, at MOI = 0.01. On Day 6 post-infection, supernatants were removed and MDM were exposed to AZT at serially diluted concentrations as indicated. The inhibition rates calculated by relative p24 production were shown for the time points of Day 8, 12 and 16 post-infection, respectively. Data were shown as mean ± SEM, donor n = 3.</p