Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) is characterized by poor prognosis after chemotherapy. Recent clinical trials have indicated, however, that allogeneic but not autologous hematopoietic stem cell transplantation (HSCT) for ATL can yield better clinical outcomes. In the present study, we investigated cellular immune responses of ATL patients who obtained complete remission after nonmyeloablative allogeneic peripheral blood HSCT from HLA-identical sibling donors. In the culture of peripheral blood mononuclear cells (PBMCs) from a post-HSCT but not pre-HSCT ATL patient, CD8⁺ CTLs proliferated vigorously in response to stimulation with autologous HTLV-I-infected T cells that had been established before HSCT in vitro. These CTLs contained a large number of monospecific CTL population directed to a HLA-A2-restricted HTLV-I Tax 11-19 epitope. The frequency of Tax 11-19-specific CD8+ CTLs in this patient markedly increased also in vivo after HSCT, as determined by staining with HLA-A2/Tax 11-19 tetramers. Similar clonal expansion of HTLV-I Tax-specific CTLs exclusively directed to a HLA-A24-restricted Tax 301-309 epitope was observed in the PBMCs from another ATL patient after HSCT from a HTLV-I-negative donor. Among four post-HSCT ATL patients tested, HTLV-I-specific CTLs were induced in the PBMC culture from three patients but not from the remaining one who had later recurrence of ATL. These observations suggested that reconstituted immunity against antigen presentation in ATL patients after HSCT resulted in strong and selective graft-versus-HTLV-I response, which might contribute to graft-versus-leukemia effects.

Background: Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs. Results: ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs. Conclusions: IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL.

Small interfering RNA (siRNA) could provide a new therapeutic approach to treating human immunodeficiency virus type 1 (HIV-1) infection. For long-term suppression of HIV-1, emergence of siRNA escape variants must be controlled. Here, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNA) corresponding to conserved target sequences within the integrase (int) and the attachment site (att) genes, both of which are essential for HIV-1 integration. Compared to shRNA targeting of the HIV-1 transcription factortat(shTat), shRNA againstint(shIN) or the U3 region ofatt(shU3) showed a more potent inhibitory effect on HIV-1 replication in human CD4⁺T cells. Infection with a high dose of HIV-1 resulted in the emergence of escape mutants during long-term culture. Of note, limited genetic variation was observed in the viruses resistant to shIN. A combination of shINs against wild-type and escape mutant sequences had a negative effect on their antiviral activities, indicating a potentially detrimental effect when administering multiple shRNA targeting the same region to combat HIV-1 variants. The combination of shIN and shU3attexhibited the strongest anti-HIV-1 activity, as seen by complete abrogation of viral DNA synthesis and viral integration. In addition, a modified long-hairpin RNA spanning the 50 nucleotides in the shIN target region effectively suppressed wild-type and shIN-resistant mutant HIV-1. These results suggest that targeting of incoming viral RNA before proviral DNA formation occurs through the use of nonoverlapping multiple siRNAs is a potent approach to achieving sustained, efficient suppression of highly mutable viruses, such as HIV-1.

Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.

Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.

There has been accumulating evidence for the involvement of retroviral integrase (IN) in the reverse transcription of viral RNA. We previously identified a host factor, survival motor neuron-interacting protein 1 (SIP1/Gemin2) that binds to human immunodeficiency virus type 1 (HIV-1) IN and supports HIV-1 infection apparently at reverse transcription step. Here, we demonstrated that HIV-1 IN together with SIP1 augments reverse transcriptase (RT) activity by enhancing the assembly of RT on viral RNA in vitro. Synthetic peptides corresponding to the binding motifs within IN that inhibited the IN-SIP1 interaction abrogated reverse transcription in vitro and in vivo. Furthermore, knockdown of SIP1 reduced intracellular stability and multimer formation of IN through proteasome-mediated degradation machinery. Taken together, SIP1 appears to stabilize functional multimer forms of IN, thereby promoting the assembly of IN and RT on viral RNA to allow efficient reverse transcription, which is a prerequisite for efficient HIV-1 infection.