Masanori Kameoka

RNA Interference Directed against Poly(ADP-Ribose) Polymerase 1 Efficiently Suppresses Human Immunodeficiency Virus Type 1 Replication in Human Cells

ABSTRACT We established small interfering RNA (siRNA) directed against poly(ADP-ribose) polymerase 1 (PARP-1) that effectively reduces the expression of PARP-1 in two human cell lines. Established siRNA against PARP-1 significantly suppressed human immunodeficiency virus type 1 (HIV-1) replication, as well as the activation of the integrated HIV-1 long terminal repeat promoter. These results indicate that PARP-1 is required for efficient HIV-1 replication in human cells. We propose that PARP-1 may serve as a cellular target for RNA interference-mediated gene silencing to inhibit HIV-1 replication.

Amplification of superoxide anion generation in phagocytic cells by HIV‐1 infection

Amplification of superoxide (O− 2) generation by HIV‐1 infection was examined in two human myeloid‐monocytic cell lines. The level of O− 2 generation in HL‐60 after infection became significantly higher than that of the steady‐state. A similar phenomenon was also shown in U937, but only after acquisition of O− 2 generation ability by differentiation to macrophages. By means of the NADPH oxidase‐coupled response in infected cells, we reconstituted the O− 2‐generating machinery in cell‐free system. The results suggested that cytosolic factor(s) exerted by infection might be responsible for the amplification of O− 2 generation. Thus, HIV‐1 infection could elevate the level of oxidative stress in macrophages which might play an important role in disease progression.

Cytotoxic T lymphocyte response in mice induced by a recombinant BCG vaccination which produces an extracellular α antigen that fused with the human immunodeficiency virus type 1 envelope immunodominant domain in the V3 loop

The host immune response of cell-mediated immunity, particularly that of cytotoxic T lymphocytes (CTLs), is a major immune defence mechanism which may provide resistance to a human immunodeficiency virus type 1 (HIV-1) spread leading to acquired immune deficiency syndrome (AIDS). To prevent the accompanying activity of HIV-1 proteins responsible for the loss of helper T-lymphocyte function, it is crucial to develop a live attenuated recombinant vaccine expressing only T- or both T- and B-cell epitopes. Here, we examined the expression of the HIV-1 Env protein V3 region (15 amino acids from Arg315 to Lys329) in Mycobacterium bovis BCG as a fused form with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this recombinant BCG (rBCG), rapidly induced V3 peptide-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d. A similar CTL response was also elicited after Balb/c mice were immunized with the same rBCG even when pre-inoculated with non-recombinant BCG. Thus, the rapid induction of HIV-1-specific CTLs indicates that this vaccine may be a therapeutic approach to preventing progression to AIDS.

The Tat Protein of Human Immunodeficiency Virus Type 1 (HIV-1) Can Promote Placement of tRNA Primer onto Viral RNA and Suppress Later DNA Polymerization in HIV-1 Reverse Transcription

ABSTRACT Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.

Role for Human Immunodeficiency Virus Type 1 Tat Protein in Suppression of Viral Reverse Transcriptase Activity during Late Stages of Viral Replication

ABSTRACT We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.

Two N-Linked Glycosylation Sites in the V2 and C2 Regions of Human Immunodeficiency Virus Type 1 CRF01_AE Envelope Glycoprotein gp120 Regulate Viral Neutralization Susceptibility to the Human Monoclonal Antibody Specific for the CD4 Binding Domain

ABSTRACT A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses.

Superoxide enhances the spread of HIV-1 infection by cell-to-cell transmission

Oxidative stress is thought to be involved in the progression of human immunodeficiency virus type 1 (HIV‐1)‐induced disease. We examined the effect of Superoxide (O2 −) on HIV‐1 spread in cultured human CD4+ cell lines. The O2 − significantly enhanced cell‐to‐cell transmission of HIV‐1, although its effect on HIV‐1 replication was not evident, presumably due to its cytostatic activity. The effect was notable on the HIV‐1 transmission from macrophages to T lymphocytes by endogenous, macrophage‐generated O2 −. This amplification was specifically reduced to the steady‐state level by antioxidants, and further to the basal level by anti‐CD4 antibodies, indicating the specificity of O2 − for enhancing HIV‐1 spread by cell‐to‐cell transmission.

A chain section containing epitopes for cytotoxic T, B and helper T cells within a highly conserved region found in the human immunodeficiency virus type 1 Gag protein

Cell-mediated immune responses constitute a major defense against the spread of human immunodeficiency virus type 1 (HIV-1). However, multiple alterations within a particular epitope may accumulate during disease progression, allowing the virus to escape cytotoxic T lymphocytes (CTLs). Therefore, the best candidate for a peptide vaccine that would prevent the onset of the disease might be a chain section containing epitopes for the generation of CTLs in regions of conserved sequences among different HIV-1 isolates. We previously showed that immunizing mice with synthetic peptides consisting of 23-amino acids (Gag-23mer; 287-309 amino acid residues) in a highly conserved region derived from the major core protein p24 of HIV-1 generates specific CTLs as well as antibodies. Here, we identified one CTL (T-1; 291-300) and two B-cell (B-1; 290-299 and B-2; 300-309) epitopes, all of which consisted of 10 amino acids within the region. In addition, helper T cells primed by the Gag-23mer peptide were proliferated by in vitro stimulation with a 21mer (H-1; 289-309) or a 19mer (H-2; 291-309) peptide, but not with a 17mer peptide (293-309) or 19mer peptide (287-305). Immunization with the H-1 peptide generated an antibody reactive to B-1, but not B-2, whereas that with H-2 generated an antibody reactive to B-2, but not B-1. CTLs were not generated by immunization with these peptides, indicating that the entire sequence of Gag-23mer is the helper epitope for CTLs. Thus, the Gag-23mer is a chain section containing epitopes for cytotoxic T, B and helper T-cells within a highly conserved region of HIV-1 Gag protein.

Poly (I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells

BackgroundDouble-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C)], are recognized by toll-like receptor 3 (TLR3) and induce interferon (IFN)-β in many cell types. Poly (I:C) is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C) elicited IFN-α/β production and natural killer (NK) cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV) infection, the viruses are cleared within 7–10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance.ResultsThe effects of Poly (I:C) on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C) suppressed cytopathic effects (CPE) induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C) and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C)-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C) treatment of BEAS-2B cells induced the production of IFN-β and increased the expression of anti-viral genes, including IFN-α, IFN-β, MxA, and OAS. Both Poly (I:C) and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells.ConclusionsCHIKV is sensitive to innate immune response induced by Poly (I:C). The inhibition of CHIKV replication by Poly (I:C) may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.

Genotypic characterization of CRF01_AE env genes derived from human immunodeficiency virus type 1-infected patients residing in Central Thailand.

CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. HIV-1 env genes were amplified by polymerase chain reaction from blood samples of HIV-1-infected patients residing in Thailand in 2006, and cloned into the pNL4-3-derived reporter viral construct. Generated envelope protein (Env)-recombinant virus was examined for its infectivity, and then 35 infectious CRF01_AE Env-recombinant viruses were selected. Sequencing analysis revealed that the interclone variation of the deduced amino acid sequences was higher in CRF01_AE env genes isolated in 2006 than in those isolated in the early 1990s, suggesting that env gene variation has been increasing gradually among CRF01_AE viruses prevalent in Thailand. We also examined the characteristics of the deduced amino acid sequences of 35 CRF01_AE env genes. Our results may provide useful information to help in better understanding the genotype of env genes of CRF01_AE viruses currently circulating in Thailand.