CNI-H0294, a nuclear importation inhibitor of the human immunodeficiency virus type 1 genome, abrogates virus replication in infected activated peripheral blood mononuclear cells

Abstract

Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin alpha. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin alpha and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

FIG. 1

Binding of the HIV-1 preintegration complex to human karyopherin α. Jurkat cells were infected with HIV-1_LAI (200 ng of p24/10⁶ cells). After a 20-h infection period the cells were solubilized and the cell lysates were preincubated with increasing concentrations of CNI-H0294, as indicated, prior to the addition of gst-hSRP1α. The PIC-gst-hSRP1α complexes were sedimented with glutathione-coated beads. The levels of HIV-1 DNA in the sedimented fractions were evaluated by PCR.

FIG. 2

CNI-H0294 inhibits 2-LTR circle formation in infected cells. Anti-CD3 MAb-activated PBMC cultures (a) or Jurkat cell cultures (b) were preincubated with CNI-H0294 and were infected with either HIV-1_M1 (20 ng of p24/10⁶ cells) for 40 h (a) or HIV-1_LAI (50 ng of p24/10⁶ cells) for 20 h (b) in the presence of the compound. The presence of 2-LTR circle forms of the HIV-1 DNA was a measure of nuclear translocation, while PCR analysis of gag sequences was used as a measure of the overall HIV-1 DNA content, reflecting viral entry into the cells and reverse transcription.

FIG. 3

CNI-H0294 inhibits de novo infection of activated PBMCs. PBMCs from a seronegative individual were activated with an anti-CD3 MAb in the presence of increasing concentrations of CNI-H0294, as indicated, and were then infected with HIV-1_M1 (20 ng of p24/10⁶ cells). (a) Virus production was evaluated on day 6 after activation by a p24-specific enzyme-linked immunosorbent assay; the virus DNA content in pooled cell lysates was evaluated by PCR (inset autoradiograph). (b) Cell proliferation was evaluated on day 4 after activation by measuring the level of incorporation of [³H]thymidine.

FIG. 4

CNI-H0294 limits virus replication in cultures of activated PBMCs from seropositive individuals. PBMCs collected from two seropositive individuals (PBMC culture Z78 [a and b]; PBMC culture Z44 [c and d]) were activated with anti-CD3 MAb in the presence of increasing concentrations of CNI-H0294 (as indicated). (a and c) Virus production was evaluated on day 6 after activation as described in the legend to Fig. 3. Viral DNA content (a, inset autoradiograph) in pooled cell lysates was evaluated by PCR. (b and d) Cell proliferation was evaluated on day 4 after activation.