Makoto Kubo

Identification of Critical Amino Acid Residues in Human Immunodeficiency Virus Type 1 IN Required for Efficient Proviral DNA Formation at Steps prior to Integration in Dividing and Nondividing Cells

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is thought to have several putative roles at steps prior to integration, such as reverse transcription and nuclear transport of the preintegration complex (PIC). Here, we investigated new functional aspects of HIV-1 IN in the context of the viral replication cycle through point mutagenesis of Ser, Thr, Tyr, Lys, and Arg residues conserved in IN, some of which are located at possible phosphorylation sites. Our results showed that mutations of these Ser or Thr residues had no effect on reverse transcription and nuclear transport of PIC but had a slight effect on integration. Of note, mutations in the conserved KRK motif (amino acids 186 to 189), proposed previously as a putative nuclear localization signal (NLS) of HIV-1 IN, did not affect the karyophilic property of HIV-1 IN as shown by using a green fluorescent protein fusion protein expression system. Instead, these KRK mutations resulted in an almost complete lack of viral gene expression due to the failure to complete reverse transcription. This defect was complemented by supplying wild-type IN in trans, suggesting a trans-acting function of the KRK motif of IN in reverse transcription. Mutation at the conserved Tyr 143 (Y143G) resulted in partial impairment of completion of reverse transcription in monocyte-derived macrophages (MDM) but not in rhabdomyosarcoma cells. Similar effects were obtained by introducing a stop codon in the vpr gene (DeltaVpr), and additive effects of both mutations (Y143G plus DeltaVpr) were observed. In addition, these mutants did not produce two-long terminal repeat DNA, a surrogate marker for nuclear entry, in MDM. Thus, the possible impairment of Y143G might occur during the nuclear transport of the PIC. Taken together, our results identified new functional aspects of the conserved residues in HIV-1 IN: i) the KRK motif might have a role in efficient reverse transcription in both dividing and nondividing cells but not in the NLS function; ii) Y143 might be an important residue for maintaining efficient proviral DNA formation in nondividing cells.

Initiation of Antiretroviral Therapy 48 Hours after Infection with Simian Immunodeficiency Virus Potently Suppresses Acute-Phase Viremia and Blocks the Massive Loss of Memory CD4+ T Cells but Fails To Prevent Disease

ABSTRACT We investigated whether a 28-day course of potent antiretroviral therapy, initiated at a time point (48 h postinoculation) following simian immunodeficiency virus (SIV) inoculation when the acquisition of a viral infection was virtually assured, would sufficiently sensitize the immune system and result in controlled virus replication when treatment was stopped. The administration of tenofovir 48 h after SIV inoculation to six Mamu-A*01-negative rhesus macaques did, in fact, potently suppress virus replication in all of the treated rhesus macaques, but plasma viral RNA rapidly became detectable in all six animals following its cessation. Unexpectedly, the viral set points in the treated monkeys became established at two distinct levels. Three controller macaques had chronic phase virus loads in the range of 1 × 103 RNA copies/ml, whereas three noncontroller animals had set points of 2 × 105 to 8 × 105 RNA copies/ml. All of the noncontroller monkeys died with symptoms of immunodeficiency by week 60 postinfection, whereas two of the three controller animals were alive at week 80. Interestingly, the three controller macaques each carried major histocompatibility complex class I alleles that previously were reported to confer protection against SIV, and two of these animals generated cytotoxic T-lymphocyte escape viral variants during the course of their infections.

Suppression of HIV-1 replication by HIV-1-irrelevant CD8+ cytotoxic T lymphocytes resulting in preservation of persistently HIV-1-infected cells in vitro.

CD8+ cells of asymptomatic HIV-1 carriers (AC) contain HIV-1-specific cytotoxic T lymphocytes (CTLs) but suppress HIV-1 replication in a class I major histocompatibility complex (MHC-I)-unrestricted manner. In order to selectively investigate the HIV-1-suppressive function of CTLs apart from HIV-1-specific cytotoxicity, HIV-1-irrelevant allo-specific CTLs were established from an HIV-1-uninfected individual and their HIV-1-suppressive activity against autologous CD4+ peripheral blood mononuclear cells (PBMC) was examined. We found that these CTLs significantly suppressed both R5 and X4-HIV-1 replication in either acutely or persistently infected autologous PBMC. Although these CTLs partially killed HIV-1-infected PBMC through Fas ligand, CTLs still suppressed late steps of HIV-1 replication in the presence of neutralizing antibodies to Fas ligand. HIV-1 replication in PBMC that had been suppressed by CTLs was reversible following depletion of CTLs from culture, analogous to the previous observation for CD8+ cell-depleted PBMC of AC. Induction of HIV-1 replication by CTL-depletion was amplified by addition of newly prepared CD4+ cells or activation with staphylococcal enterotoxin B. Our results indicate that CTLs can suppress HIV-1 replication in PBMC in an antigen-nonspecific manner and preserve infected cells in a state capable of restarting HIV-1 replication and transmission.

Role of class I major histocompatibility complex-restricted and -unrestricted suppression of human immunodeficiency virus type 1 replication by CD8+ T lymphocytes.

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (ACs) are capable of suppressing HIV-1 replication in CD4+ peripheral blood mononuclear cells (PBMC) by a variety of known and unknown mechanisms. In the present study, cell contact-dependent, major histocompatibility complex type I (MHC I)-unrestricted, CD8+ cell-mediated suppression of HIV-1 LAI replication was detected. CD8+ PBMC of ACs suppressed HIV-1 replication more efficiently in MHC I-matched CD4+ PBMC than in mismatched cells. However, even when MHC I was totally mismatched, CD8+ cells still suppressed replication to a considerable extent in CD4+ PBMC. This MHC I-unrestricted, CD8+ cell-mediated HIV-1 suppression required cell contact and was not effective against cells of the established T cell line ILT-KK. In contrast, MHC I-restricted HIV-1 suppression by CD8+ T cells was detected when ILT-KK cells were used as a target. By using these systems, we examined MHC I-restricted and -unrestricted suppressive activities of CD8+ cells in various donors in more detail. Although both types of CD8+ cell-mediated HIV-1 suppression diminished at the advanced stage of the infection, MHC I-unrestricted suppression diminished earlier than MHC I-restricted suppression, in parallel with the decline in CD4+ T cells. These results suggest that suppression by the MHC I-restricted mechanism alone may fail to protect against CD4+ T-cell loss at the late stage of infection.

Correlation of PU.1 and signal regulatory protein α1 expression in PU.1 transgenic K562 cells

PU.1 is a key transcription factor for hematopoiesis and the reduction of this protein expression plays important roles in various hematological malignancies. To identify PU.1 downstream target genes, we recently reported dual microarray analyses, using PU.1 knockdown K562 (K562PU.1KD) cells stably expressing short inhibitory RNAs versus control cells and PU.1-overexpressing K562 (K562PU.1OE) cells versus control cells. Several PU.1 candidate target genes, including cell surface receptor, signal regulatory protein (SIRP) α1, were identified. In this study, we revealed that the expression of SIRPα1 is positively correlated with the expression of PU.1 in various K562PU.1KD and K562PU.1OE cells, shown by real-time PCR and flow cytometry analyses. SIRPα1 is a negative regulator of signaling and its reduced expression is considered to play a role in the pathogenesis of hematological malignancies through the activation of downstream signaling pathways. By comparing 3 different clones of K562PU.1KD cells to their controls, we found constitutive phosphorylation of the extracellular signal-regulated kinase (ERK), but not of Akt, in these cells. Taken together, the down-regulation of PU.1 expression suppresses the expression of SIRPα1, which may play a role in the aberrant activation of ERK in these cells.

Allo-Antigen Stimulated CD8+ T-Cells Suppress NF-κB and Ets-1 DNA Binding Activity, and Inhibit Phosphorylated NF-κB p65 Nuclear Localization in CD4+ T-cells

CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).