Atsushi Koito

Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection.

Trypstatin, a new cellular Kunitz‐type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV‐1)‐infected CCRF‐CEM and uninfected Molt‐4 clone 8 at a concentration of 1 μM. Anti‐rat tongue mast cell tryptase antibodies reacted with Molt‐4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV‐1 suggest that a tryptase‐like cellular enzyme(s) is involved in HIV‐1 infection.

Preliminary evidence for inhibitory effect of glycyrrhizin on HIV replication in patients with AIDS.

Glycyrrhizin (GL) at a dose of 400-1600 mg/day (7.2-30.8 mg/kg/day) was administered intravenously for a period of more than a month, on 6 separate occasions, to 3 hemophiliacs with acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus type 1 (HIV-1) p24 antigen was detected at the beginning of 5 of the 6 treatment courses. Viral antigen was not detected at the end of or during 3 of the 5 treatment courses and decreased to a low level following the 2 other courses. These findings suggest that GL might inhibit HIV-1 replication in vivo.

APOBEC3G Generates Nonsense Mutations in Human T-Cell Leukemia Virus Type 1 Proviral Genomes In Vivo

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) induces cell proliferation after infection, leading to efficient transmission by cell-to-cell contact. After a long latent period, a fraction of carriers develop adult T-cell leukemia (ATL). Genetic changes in the tax gene in ATL cells were reported in about 10% of ATL cases. To determine genetic changes that may occur throughout the provirus, we determined the entire sequence of the HTLV-1 provirus in 60 ATL cases. Abortive genetic changes, including deletions, insertions, and nonsense mutations, were frequent in all viral genes except the HBZ gene, which is transcribed from the minus strand of the virus. G-to-A base substitutions were the most frequent mutations in ATL cells. The sequence context of G-to-A mutations was in accordance with the preferred target sequence of human APOBEC3G (hA3G). The target sequences of hA3G were less frequent in the plus strand of the HBZ coding region than in other coding regions of the HTLV-1 provirus. Nonsense mutations in viral genes including tax were also observed in proviruses from asymptomatic carriers, indicating that these mutations were generated during reverse transcription and prior to oncogenesis. The fact that hA3G targets the minus strand during reverse transcription explains why the HBZ gene is not susceptible to such nonsense mutations. HTLV-1-infected cells likely take advantage of hA3G to escape from the host immune system by losing expression of viral proteins.

Recurrent HIV-1 integration at the BACH2 locus in resting CD4+ T cell populations during effective highly active antiretroviral therapy

The persistence of latent human immunodeficiency virus type 1 (HIV-1) has been considered one of the major obstacles for eradication of the virus in infected individuals receiving successful antiretroviral therapy. To determine the contribution of integration sites to viral latency within clinical settings, an inverse polymerase chain reaction method was used to analyze integration sites in CD4(+) T cells from patients showing long-term undetectable plasma viral RNA. Of 457 sites identified in 7 patients, almost all (96%) resided within transcriptional units, usually in introns of the human genome. Studies of 18 genes in which HIV-1 integrates found them to be actively expressed in resting CD4(+) T cells. On the other hand, integration sites in the alpha satellite region was also identified in some patients, albeit at low frequency. Of particular interest, HIV-1-infected cells with multiple identical integration sites were detected in longitudinal analysis of samples from 3 patients, suggesting that these cells persist for long periods and that clonal expansion may occur. Furthermore, strong integration clusters in the BACH2 gene were observed in 2 patients (31% in patient 1 and 5% in patient 3). Our findings not only raise the possibility of biased target-site integration but also provide mechanistic insights into the long-term persistence of HIV-1.

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases

Over 40% of the human genome is recognizable as having been derived from ancient retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, with an additional few percent classified as DNA transposable elements. Endogenous retroviruses are long terminal repeat (LTR)-type retroelements that account for ~8% of human genomic DNA. Non-LTR members are present at extremely high copy numbers, with ~17% of the human genome consisting of long interspersed nuclear elements (LINEs). These LINEs modify vertebrate genomes not only through insertions, but also by the indirect replication of non-autonomous retrotransposons, such as short interspersed nuclear elements. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that confer beneficial genetic diversity and those that cause deleterious gene disruptions. The mammalian cytidine deaminases encoded by the APOBEC3 genes can restrict a broad number of exogenous pathogens, such as exogenous retroviruses, and the mobility of endogenous retroelements. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the cytidine (C) to uridine (U) deamination of apolipoprotein B (apoB) mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian apoB mRNA-editing, catalytic polypeptide (APOBEC) cytidine deaminases, which can bind to single-stranded DNA (ssDNA) as well as RNA, are able to insert mutations into ssDNA and/or RNA as a result of their ability to deaminate C to U. While these APOBEC cytidine deaminases with DNA mutagenic activity can be deleterious to cells, their biological modifications, such as protein–protein interactions and subcellular localization, in addition to their ability to bind to RNA, appear to have conferred a role for APOBECs as a cellular defense system against retroviruses and retroelements. In support of this notion, the expansion of the single APOBEC3 gene in mice to the seven APOBEC3 genes found in primates apparently correlates with the significant enhancement of the restriction of endogenous retroelements seen in primates, including humans. This review discusses the current understanding of the mechanism of action of APOBEC cytidine deaminases and attempts to summarize their roles in controlling retrotransposons.

Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C–U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patients erythrocytes. When the patients serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

The antiretroviral potency of APOBEC1 deaminase from small animal species

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Δvif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.

Impact of V2 Mutations on Escape from a Potent Neutralizing Anti-V3 Monoclonal Antibody during In Vitro Selection of a Primary Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.