Takayoshi Koyama

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80–100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

Retroelements versus APOBEC3 family members: No great escape from the magnificent seven

Retroelements comprise a large and successful family of transposable genetic elements that, through intensive infiltration, have shaped the genomes of humans and other mammals over millions of years. In fact, retrotransposons now account for approximately 45% of the human genome. Because of their genomic mobility called retrotransposition, some retroelements can cause genetic diseases; such retrotransposition events occur not only in germ cells but also in somatic cells, posing a threat to genomic stability throughout all cellular populations. In response, mammals have developed intrinsic immunity mechanisms that provide resistance against the deleterious effects of retrotransposition. Among these, seven members of the APOBEC3 (A3) family of cytidine deaminases serve as highly active, intrinsic, antiretroviral host factors. Certain A3 proteins effectively counteract infections of retroviruses such as HIV-1, as well as those of other virus families, while also blocking the transposition of retroelements. Based on their preferential expression in the germ cells, in which retrotransposons may be active, it is likely that A3 proteins were acquired through mammalian evolution primarily to inhibit retrotransposition and thereby maintain genomic stability in these cells. This review summarizes the recent advances in our understanding of the interplay between the retroelements currently active in the human genome and the anti-retroelement A3 proteins.

MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING (really interesting new gene)-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins, including major histocompatibility complex (MHC)-II, CD86, interleukin (IL)-1 receptor accessory protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 and the transferrin receptor. However, its physiological roles remain largely unknown. Here we identify MARCH8 as a novel antiviral factor. The ectopic expression of MARCH8 in virus-producing cells does not affect levels of lentivirus production, but it does markedly reduce viral infectivity. MARCH8 blocks the incorporation of HIV-1 envelope glycoprotein into virus particles by downregulating it from the cell surface, probably through their interaction, resulting in a substantial reduction in the efficiency of viral entry. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein is even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Notably, the endogenous expression of MARCH8 is high in monocyte-derived macrophages and dendritic cells, and MARCH8 knockdown or knockout in macrophages significantly increases the infectivity of virions produced by these cells. Our findings thus indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells, and that it is a potent antiviral protein that targets viral envelope glycoproteins and reduces their incorporation into virions.

DNA damage enhances integration of HIV-1 into macrophages by overcoming integrase inhibition

BackgroundThe prevention of persistent human immunodeficiency virus type 1 (HIV-1) infection requires the clarification of the mode of viral transduction into resting macrophages. Recently, DNA double-strand breaks (DSBs) were shown to enhance infection by D64A virus, which has a defective integrase catalytic activity (IN-CA). However, the mechanism by which DSBs upregulate viral transduction was unclear. Here we analyzed the roles of DSBs during IN-CA–independent viral transduction into macrophages.ResultsWe used cellular systems with rare-cutting endonucleases and found that D64A virus integrated efficiently into the sites of artificially induced DSBs. This IN-CA-independent viral transduction was blocked by an inhibitor of ataxia telangiectasia mutated protein (ATM) but was resistant to raltegravir (RAL), an inhibitor of integrase activity during strand transfer. Moreover, Vpr, an accessory gene product of HIV-1, induced DSBs in resting macrophages and significantly enhanced the rate of IN-CA-independent viral transduction into macrophages with concomitant production of secondary viruses.ConclusionDSBs contribute to the IN-CA–independent viral infection of macrophages, which is resistant to RAL. Thus, the ATM-dependent cellular pathway and Vpr-induced DNA damage are novel targets for preventing persistent HIV-1 infection.

Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide

Active targeting by monoclonal antibodies (mAbs) combined with nanosize superparamagnetic iron oxide (SPIO) is a promising technology for magnetic resonance imaging (MRI) diagnosis. However, the clinical applicability of this technology has not been investigated using appropriate controls. It is important to evaluate the targeting technology using widely used clinical 1.5-Tesla MRI in addition to the high-Tesla experimental MRI. In this study, we measured mAb-conjugated dextran-coated SPIO nanoparticles (CMDM) in vivo using clinical 1.5-Tesla MRI. MRI of tumor-bearing mice was performed using a simple comparison between positive and negative tumors derived from the same genetic background in each mouse. The system provided significant tumor-targeting specificity of the target tumor. To the best of our knowledge, this is the first report on the specific detection of target tumors by mAb-conjugated SPIO using clinical 1.5-Tesla MRI. Our observations provide clues for reliable active targeting using mAb-conjugated SPIO in clinical applications.

APOBEC3G oligomerization is associated with the inhibition of both Alu and LINE-1 retrotransposition.

Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A–H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

Identification of SNF2h, a Chromatin-Remodeling Factor, as a Novel Binding Protein of Vpr of Human Immunodeficiency Virus Type 1

Vpr, an accessory gene of human immunodeficiency virus type 1, encodes a virion-associated nuclear protein that plays an important role in the primary viral infection of resting macrophages. It has a variety of biological functions, including roles in a cell cycle abnormality at G2/M phase, apoptosis, nuclear transfer of preintegration complex, and DNA double-strand breaks (DSBs), some of which depend on its association with the chromatin of the host cells. Given that DSB signals are postulated to be a positive factor in the viral infection, understanding the mode of chromatin recruitment of Vpr is important. Here, we identified SNF2h, a chromatin-remodeling factor, as a novel binding partner of Vpr involved in its chromatin recruitment. When endogenous SNF2h protein was extensively downregulated by SNF2h small interfering RNA (siRNA), the amount of Vpr loaded on chromatin decreased to about 30% of the control level. Biochemical analysis using a mutant Vpr suggested that Vpr binds SNF2h via HFRIG (amino acids 71–75 depicted by single letters) and the Vpr mutant lacking this motif lost the activity to induce DSB-dependent signals. Consistently, Vpr-induced DSBs were attenuated by extensive downregulaion of endogenous SNF2h. Based on these data, we discuss the role of DSB and DSB signals in the viral infection.

Membrane-associated RING-CH (MARCH) 8 protein inhibits HIV-1 infection

Background The MARCH (membrane-associated RING-CH) 8 protein is a member of a recently discovered MARCH family of RING-finger E3 ubiquitin ligases, 11 members of which have been identified. MARCH8 downregulates several transmembrane proteins, such as MHC-II, CD86, TRAIL receptor 1, and CD98. We have recently reported that MARCH8 also negatively regulates cell-surface expression of transferrin receptor [1]. During the preparation of MARCH8-encoding lentiviral vector, we observed that the infectivity of the vector was considerably lower than that of the control vector, suggesting that MARCH8 might have the ability to reduce viral infectivity. Here, we have assessed the impact of MARCH8 expression on HIV-1 replication.