A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system.

Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cytidine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were performed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells.

Pathogens including autoantigens all failed to induce systemic lupus erythematosus (SLE). We, instead, studied the integrity of host's immune response that recognized pathogen. By stimulating TCR with an antigen repeatedly to levels that surpass host's steady-state response, self-organized criticality, SLE was induced in mice normally not prone to autoimmunity, wherein T follicular helper (Tfh) cells expressing the guanine nucleotide exchange factor DOCK8 on the cell surface were newly generated. DOCK8+Tfh cells passed through TCR re-revision and induced varieties of autoantibody and lupus lesions. They existed in splenic red pulp and peripheral blood of active lupus patients, which subsequently declined after therapy. Autoantibodies and disease were healed by anti-DOCK8 antibody in the mice including SLE-model (NZBxNZW) F1 mice. Thus, DOCK8+Tfh cells generated after repeated TCR stimulation by immunogenic form of pathogen, either exogenous or endogenous, in combination with HLA to levels that surpass system's self-organized criticality, cause SLE.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins (APOBEC3s) are deaminases that convert cytosines to uracils predominantly on a single-stranded DNA, and function as intrinsic restriction factors in the innate immune system to suppress replication of viruses (including retroviruses) and movement of retrotransposons. Enzymatic activity is supposed to be essential for the APOBEC3 antiviral function. However, it is not the only way that APOBEC3s exert their biological function. Since the discovery of human APOBEC3G as a restriction factor for HIV-1, the deaminase-independent mode of action has been observed. At present, it is apparent that both the deaminase-dependent and -independent pathways are tightly involved not only in combating viruses but also in human tumorigenesis. Although the deaminase-dependent pathway has been extensively characterized so far, understanding of the deaminase-independent pathway remains immature. Here, we review existing knowledge regarding the deaminase-independent antiretroviral functions of APOBEC3s and their molecular mechanisms. We also discuss the possible unidentified molecular mechanism for the deaminase-independent antiretroviral function mediated by mouse APOBEC3.

Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.

CD8(+) tissue-resident memory T cells (T-RM cells) reside permanently in nonlymphoid tissues and provide a first line of protection against invading pathogens. However, the precise localization of CD8(+) T-RM cells in the lung, which physiologically consists of a markedly scant interstitium compared with other mucosa, remains unclear. In this study, we show that lung CD8(+) T-RM cells localize predominantly in specific niches created at the site of regeneration after tissue injury, whereas peripheral tissue-circulating CD8(+) effector memory T cells (T-EM cells) are widely but sparsely distributed in unaffected areas. Although CD69 inhibited sphingosine 1-phosphate receptor 1-mediated egress of CD8(+) T cells immediately after their recruitment into lung tissues, such inhibition was not required for the retention of cells in the T-RM niches. Furthermore, despite rigid segregation of T-EM cells from the T-RM niche, prime-pull strategy with cognate antigen enabled the conversion from T-EM cells to T-RM cells by creating de novo T-RM niches. Such damage site-specific localization of CD8(+) T-RM cells may be important for efficient protection against secondary infections by respiratory pathogens.

A hybrid comprising an autophagy-inducing peptide (AIP) and a cell-penetrating peptide (CPP) connected via heterodimeric leucine zippers was generated and delivered into cells. The hybrid successfully induced autophagy without significant cell death, while the same AIP directly connected to a CPP caused both autophagy and significant cell death.

Background: HIV-1 Vpr-mediated G(2) cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G(2) arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown. Methods: Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response. Results: We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX. Conclusions: The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the interaction with DDB1. The ability of SIVagm Vpr to associate with DDB1, but not DCAF1, can explain the species-specificity of SIVagm Vpr-mediated G(2) arrest.

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta-and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Delta 5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Delta 5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV-1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply because of the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation.

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). To develop a better animal model for the investigation of HTLV-1 infection, we established a transgenic (Tg) rat carrying the human CRM1 (hCRM1) gene, which encodes a viral RNA transporter that is a species-specific restriction factor. At first we found that CRM1 expression is elaborately regulated through a pathway involving protein kinase C during lymphocyte activation, initially by posttranscriptional and subsequently by transcriptional mechanisms. This fact led us to use an hCRM1-containing bacterial artificial chromosome clone, which would harbor the entire regulatory and coding regions of the CRM1 gene. The Tg rats expressed hCRM1 protein in a manner similar to expression of intrinsic rat CRM1 in various organs. HTLV-1-infected T-cell lines derived from these Tg rats produced 100- to 10,000-fold more HTLV-1 than did T cells from wild-type rats, and the absolute levels of HTLV-1 were similar to those produced by human T cells. We also observed enhancement of the dissemination of HTLV-1 to the thymus in the Tg rats after intraperitoneal inoculation, although the proviral loads were low in both wild-type and Tg rats. These results support the essential role of hCRM1 in proper HTLV-1 replication and suggest the importance of this Tg rat as an animal model for HTLV-1.

The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G(2) arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G(2) arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.

APOBEC3 proteins comprise a multigene family of antiviral cytidine deaminases that are active against human immunodeficiency virus, simian immunodeficiency virus, endogenous retroelements. The Vif protein of lentiviruses binds to specific APOBEC3 proteins, notably A3F and A3G, to induce their degradation by proteasomes. APOBEC3 proteins are of two types, those with a single deaminase domain such as human (h)A3A and hA3C and those with two cytidine deaminase domains (CDD) such as hA3G, hA3F, hA3B and the mouse APOBEC3, mA3. In hA3G, both active sites are required for antiviral function but serve separate functions. CDD2 mediates the C to U deamination of the human immunodeficiency virus type 1 genome, whereas CDD1 binds the viral RNA to allow for virion packaging. Here we analyzed the role of the two domains in additional APOBEC3 family members. We analyzed APOBEC3 proteins in which either the critical glutamic acid residue or the Zn2+ coordination amino acid residues in the active sites were mutated. The separation of function of the domains is maintained in hA3B and hA3F, but in the mouse protein mA3, the roles of the two domains are reversed. Deamination is mediated by CDD1, whereas encapsidation and dimerization are mediated by CDD2. Antiviral function of each of the APOBEC3 proteins was largely attributable to deaminase activity. Deaminase-independent antiviral activity of the active site mutants was minor. These findings suggest that the two active sites have different functions but that these functions can be interchanged in different APOBEC3 family members.

Rat ortholog of human CRM1 has been found to be responsible for the poor activity of viral Rex protein, which is essential for RNA export of human T cell leukemia virus type 1 (HTLV-1). Here, we examined the species-specific barrier of HTLV-I by establishing rat cell lines, including both adherent and CD4(+) T cells, which express human CRM1 at physiological levels. We demonstrated that expression of human CRM1 in rat cells is not harmful to cell growth and is sufficient to restore the synthesis of the viral structural proteins, Gag and Env, at levels similar to those in human cells. Gag precursor proteins were efficiently processed to the mature forms in rat cells and released into the culture medium as sedimentable viral particles. An HTLV-1 pseudovirus infection system suggested that the released virus particles are fully infectious. Our newly developed reporter cell system revealed that Env proteins produced in rat cells are fully fusogenic, which is the basis for cell-cell HTLV-1 infection. Moreover, we show that the early steps in infection, from post-entry uncoating to integration into the host chromosomes, occur efficiently in rat cells. These results, in conjunction with reports describing efficient entry of HTLV-1 into rat cells, may indicate that HTLV-1 is unique in that its major species-specific barrier is determined by CRM1 at a viral RNA export step. These observations will enable us to construct a transgenic rat model expressing human CRM1 that is sensitive to HTLV-1 infection. (c) 2006 Elsevier SAS. All rights reserved.

Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of human T-cell leukemia virus type 1 (HTLV-1) as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA. Although there are only 24 amino acid differences between hCRM1 and rat CRM1 (rCRM1), rCRM1 can hardly support Rex activity, suggesting a role for rCRM1 as a determinant restricting the host range of HTLV-1. Here, we used a series of mutants, which were generated by interchanging residues of these CRM1s, to examine the relationship of hCRM1 functions. The functions for Rex multimerization and binding to nuclear export signals are mapped to different amino acid residues, and these are separable, suggesting that CRM1 not only functions as an export receptor but also participates in the formation of the RNA export complex through higher-ordered interaction with Rex. The region for the interaction with RanBP3, comprising four residues (amino acids [aa] 411, 414, 474, and 481), and the region for Rex multimerization, including two residues (aa 411 and 414), form an overlapped domain. Our results provide the molecular basis underlying the species-specific ability of HTLV-1 to propagate in human cells.

Rex protein is a post-transcriptional regulator that is required for the expression of unspliced and incompletely spliced viral mRNAs that encode Gag and Env proteins. Rex binds directly Rex response element (RxRE) in human T cell leukemia virus type 1 (HTLV-1) RNAs and then undergoes multimerization on the RNA, which is necessary to export viral RNAs. Human CRM1, which is cellular machinery to export proteins from the nucleus to the cytoplasm, has been found to bind to Rex directly. Therefore, Rex executes its function by bridging viral RNAs and the cellular export machinery. In the first half of this review article we describe additional roles of CRM1 in Rex functioning, such as involvement in Rex multimerization on RNA. In the latter half, we report that rat CRM1 export little viral RNA because it has poor ability to induce the Rex multimerization. Our results suggest that the poor ability of rat CRM1 as a cofactor of Rex may be responsible for the poor replication of HTLV-1 in rats. Such results provide a clue to means of improving presently incomplete rat models of HTLV-1-related diseases.

The human immunodeficiency virus type I (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced and singly spliced viral mRNAs by bridging viral RNA and export receptor human CRM1 (hCRM1). Ribonucleoprotein complex formation, including the oligomerization of Rev proteins on viral RNA, must occur to allow export. We show here that Rev-Rev interactions, which are a basis of complex formation, can be initiated without cellular factors and are subsequently enhanced by hCRM1-Ran-GTP. Furthermore, we reveal functions for the Rev carboxy-terminal (C-terminal) region, which is well conserved among many HIV-1 strains, and for which no function has been reported. This region is required for the efficient binding of Rev to hCRM1 and consequently for nuclear export, Rev-Rev dimerization, and full Rev transactivator activity. Consistent with these results, a HIV-1 proviral plasmid that expresses a C-terminally truncated Rev mutant protein produces smaller amounts of the p24 antigen than does a plasmid that possesses an intact rev gene. These results indicate the functional importance of the C-terminal region for full Rev activity, which leads to efficient HIV-1 replication.

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.

We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex protein from the nucleus to the cytoplasm. A Rex protein with a mutation in the activation domain, RexM90, lost both the ability to hind to hCRM1 and the ability to multimerize. The overexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex protein. These two functions were simultaneously restored by the overexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex protein.

In the process of identifying a host gene that regulates the production of neutralizing antibodies (Ab) upon retrovirus infection, we found genetic polymorphisms at the mouse Apobec3 locus. However, how this cytidine deaminase could affect Ab production was unclear. One hypothesis was that APOBEC3 (A3) might affect somatic hypermutation in B lymphocytes. To examine this, we established knock-in strains of mice that express A3 protein tagged with an in-frame FLAG peptide. Tagged A3 was detected in B cells, and distinctive high expression was observed in germinal center cells. Antigenic stimulation increased germinal center cells but A3 expression levels in each cell did not change. Further, in the presence of A3 processing of viral gag gene products was hampered and immature particles increased among budding virions. Thus, A3 may indirectly affect Ab production through protecting B cells from infection and increasing antigenically intact but non-replicating immature virions.

We aim to treat diseases by safely and efficiently delivering proteins and functional peptides into cells. Here, we conjugated the Nanog protein to a heterodimeric leucine zipper peptide (LzK). In addition, an cell-penetrating peptide was conjugated to LzE peptide that forms a hybrid with LzK. When these peptides were mixed, the Nanog protein was successfully delivered into the cytoplasm and further into the nucleus, and the function possessed by Nanog protein was successfully expressed.

APOBEC3 is a cytidine deaminase and inhibits the replication of various types of viruses through the deaminase-dependent and -independent ways. Understanding the entire mechanisms that underlie the anti-viral function of APOBEC3 remains incomplete. Here, we investigated how APOBEC3 interferes with the replication of human immunodeficiency virus (HIV-1) and murine leukemia viruses (MuLVs). We found that APOBEC3 binds Gag-Pol precursor polyprotein and interferes with the production of mature viral protease (PR) autoprocessed from the Gag-Pol precursor, leading to an increase in immature virus particles, which can not infect with cells. Our results suggest that this inhibitory effect of APOBEC3 is independent on its deaminase activity and probably mediated through the promotion of aberrant digestion of Gag-Pol. We also found that APOBEC3 may reduce virion production likely through the binding to Gag-Pol and subsequent inhibition of virion assembly.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication through a deamination-independent mechanism in which the reverse transcription is considered the prime target process. However, other steps in virus replication can also be targeted by mA3. We have investigated the possible effect of mA3 on viral protease-mediated processes. We found that mA3 directly bound both mature viral protease and Pr180gag-pol precursor. Furthermore, the autoprocessing of Pr180gag-pol was impeded by mA3, resulting in reduced production of mature viral protease. Exon 5-containing and -lacking isoforms of mA3 equally exhibited this antiviral activity, and physiologically expressed levels of mA3 impeded Pr180gag-pol autocatalysis and Pr65gag processing. This blockade was independent of the deaminase activity and required the C-terminal half of mA3. These results suggest that Pr180gag-pol autoprocessing can be a critical step for mA3 to exert its deaminase-independent antiretroviral activity.

We had discovered that HIV-1 Vpr has the ability to control an autophagy. Here we have tried to identify cellular factor(s) involved in this regulation and disclose its molecular mechanisms. We found that the Vpr-mediated autophagy regulation does not depend on cellular factors which have previously been reported to interact with Vpr. Interestingly, Vpr has two functions in autophagy regulation, augmentation of autophagosome formation and inhibition of autolysosome maturation. We identified a key factor by which Vpr facilitates autophagosome formation. Vpr seems to activate the cellular protein binding to the identified factor to initiate autophagy. We find that Vpr does not disturb the autolysosome formation but clearly impairs the acidity of cellular acidic compartments. These results indicate that Vpr decrease enzymatic function in acidic organelles through disruption of pH, leading to inhibition of autolysosome function.

APOBEC3 is a DNA mutator that restricts retrovirus replication, and its allelic differences have been associated with kinetics of the production of virus-neutralizing antibodies. We wished to elucidate how APOBEC3 affects antibody production. CD8+ T cells were dispensable while CD4+ T cells were crucial for the elimination of virus-infected erythroid cells. In the absence of B-lymphocytes, infected erythroid cells were eliminated but retrovirus replication persisted in myeloid cells. Friend retrovirus also infected the thymus, and thymic expression of the viral antigens lead to negative selection of virus-specif T cells. Further, mice lacking activation-induced cytidine deaminase nevertheless produced neutralizing antibodies, and non-mutated IgM conferred resistance to infection in the presence of virus antigen-primed CD4+ T cells. Thus, APOBEC3 seems to interfere with virus-induced derangement of CD4+ T-cell functions that are crucial for the production of neutralizing antibodies.

We identified a new function of human Rac2. Rac2 negatively or positively regulates several cytokines production at transcriptional level. The transcriptional regulation by Rac2 involves specific molecules acting in cellular signal transduction pathway. The regulation of inflammatory cytokines would be the one of mechanisms by which Rac2 mediates the anti-HIV-1 activity. We found that Rac2 associates with viral proteins which can control autophagy. These interactions implied that Rac2 suppresses HIV-1 replication thorough modification of cellular autophagy activity controlled by viral proteins.

Mouse APOBEC3, a host factor that restricts the replication of exogenous retrovirus, is abundantly expressed in virus-resistant C57BL/6 (B6) mice. We have found that loss of APOBEC3 causes spontaneous mammary carcinoma and T cell lymphomas in B6 mice. The transcripts of endogenous mouse mammary tumor virus are abundant in the tumor cells prepared from APOBEC3-deficient mice. These results suggest that APOBEC3 contributes to the control of activated endogenous retrovirus.

Polymorphisms at the mouse APOBEC3(mA3) gene locus have been associated with different susceptibilities to infection with mouse leukemia viruses. In virus-resistant C57BL/6(B6) mice, mA3 transcripts and protein are more abundant than those in susceptible BALB/c mice, and these strains of mice also express mA3 transcripts with different splicing patterns. B6 mice express predominantly the exon 5-deficient transcript, which confer more efficient translation than the full-length transcript. By employing in vitro splicing assays using genomic DNA clones, we identified two critical determinants that regulate exon 5 inclusion into mA3 transcripts : the number of TCCT repeats upstream of exon 5 and the newly identified single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary.

Several genetic variations have been identified as factors which affect HIV-1 infection in human. We have recently discovered that human rac2 gene is one of the restriction genes for HIV-1 infection and individuals who possess high-expression type rac2 allele in their genome tend to be more resistant to HIV-1 infection as compared to people with low-expression type rac2 gene allele. During the term of this grant, we identified a single nucleotide polymorphism controlling Rac2 expression in an intron region of the rac2 gene. We found that Rac2 decreases CCR5 expression, a critical co-receptor for HIV-1 infection, at transcriptional level but increases CCL5 which binds to CCR5 and inhibits HIV-1 infection. These results suggest that higher Rac2 expression affects the HIV-1-cell interaction, leading to more resistant against HIV-1 infection.

研究目的 近交系が確立され、遺伝子操作が可能なラットにHIVが感染できるならば、治療法と予防法の開発に大いに役立つ.既に、我々はヒトCRM1を発現させたラット上皮細胞株にHIV plasmidを導入すると、ヒト細胞に準じるHIV粒子が生産されること、Tgラット体内でhCRM1が機能することを示した。そこで今年度は受容体とhCRM1を共発現するTgラットを作成すると共に、ラットT細胞株におけるヒト因子の要求性と抑制因子をさらに検討し、同定された因子を発現するTgラットを作成することを目的とした。 研究成果 ラットT細胞でのHIV-1の増殖はhCRM1とhCyclinT1の単独発現では数倍の増強にすぎないが、共発現によって数十倍の相乗的な増強が見られた。ヒトCD4/CCR5/CRM1/CyclinT1の4種を発現するラットT細胞ではヒトmolt4CCR5の1/3〜1/10のp24が生産された。又、ラットには2種類の阻害因子があることが示唆された。hCCR5/hCXCR4を発現するTgラットを作成した。 考察 今年度、ラットT細胞株を用いてHIV-1の増殖に必用なヒト因子としてCRM1以外にCyclinT1を同定した。さらに、阻害因子の存在を示唆した。従って、HIV-1感受性のラットを作成するためには、hCRM1とhCyclinT1を発現し、阻害因子をノックダウンしたTgラットを作成する必用がある。

HTLV-1のRexはウイルスmRNAの核外輸送を担うウイルスタンパク質である。Rexの機能にはCRM1やRanBP3に代表される細胞性因子との直接または間接的な相互作用が不可欠であり、これらの細胞内での発現制御はウイルス複製に大きく影響すると考えられる。そこで本年度はCRM1を中心にそれらの発現様式を調べた。まず細胞周期に依存的なCRM1発現制御の有無を調べるため、既存の細胞周期調節試薬(ノコダゾール等)を利用した。その結果CRM1はG1,G2,M期といった細胞周期でも一定の発現量(タンパク質レベル)を保っていることが分かった。次に分化に伴うCRM1発現制御について検討した。この際HL60細胞を利用した。この細胞株はPMAの刺激によりマクロファージ様の細胞に分化することが報告されている。PMA刺激後分化を形態観察で追跡し同時に細胞中のCRM1発現量を測定したところ、分化前後において発現量に変化はなかった。この際RNAレベルでもその発現量に変化がないことが確認された。またCRM1以外にRexの機能に必要であるRanBP3やImportin-beta等の発現量もRNAレベルにおいて分化前後で変化しないことが分かった。さらにアポトーシスにおけるCRM1発現制御についても種々のアポトーシス誘導刺激試薬を利用することで検討した。その結果アポトーシス前後においてCRM1発現量の変化は確認されなかった。これらの結果から、CRM1は細胞の多様な生理反応に必須のタンパク質であり、種々の細胞刺激によらず一定量発現していることが細胞にとって必須であることが示唆された。今後はHTLV-1感染による(主にHTLV-1のTaxタンパク質による)様々な細胞へのストレスに対応した細胞性因子の発現制御があるのか調べる予定である。

今年度、エイズウイルス(HIV)がラットで増殖できない原因を追求するために、ウイルスRNAの核外輸送過程を検討した.まず、ウイルス輸送因子Revがラット細胞で機能するかどうかを2種類のレポーターを用いて調べた.1つはHIVゲノムのenv領域にCAT遺伝子を挿入したもの(pDM128)、2つ目はHIVのほぼ全長を持つものである.PDM128を用いてCATの生産量で測定するとRevは十分効率よく働くが、後者のレポーターからはGag/Envがヒト細胞の1/100程度しか生産されなかった.ウイルスRNAの核から細胞質への輸送を半定量PCR,定量PCRで測定したところ、両細胞間で差がなかった.また、Gagタンパク質は両細胞中で安定であった.この結果はウイルスRNAがヒト細胞中で効率よく翻訳されるとこを示唆している. 次に細胞輸送因子であるCRM1が原因であるかどうか調べた.ラット細胞にヒトCRM1を導入すると、Gagタンパク質が約10倍効率よく生産された.ウイルスRNAの輸送効率は変わらなかった.これらの結果は、ヒトCRM1は核から細胞質へのRNA輸送を司るだけではなく効率の良い翻訳にも関与していることを示唆している.ラットCRM1はウイルスRNAを輸送できるが、効率的な翻訳を行えない。これはCRM1の新機能の発見である。 次に、ヒトCRM1を恒常的に発現するラット細胞を作製して、HIVタンパク質と感染性子孫ウイルスが生産されるかどうか調べた.いずれもヒト細胞に準じる生産量であった.このことは、ラットでHIVが増殖できない1原因がCRM1にあることを示している.

Human T cell leukemia virus type1 (HTLV-1) replicates poorly in rats owing to a low activity of Rex in rat cells. Rat autholog of human CRM 1, a cofactor of Rex, is found to be responsible for the poor activity of Rex, because it does not support the Rex multimerization, which is essential for viral RNA export. In this Project, we intended to demonstrate that rat CRM1 is a major block against HTLV-1replication and expression of human CRM1 in rat cells is sufficient to recover viral protein synthesis. Finally we aimed to produce a transgenic rat expressing human CRM1. Then we have gotten the following results. 1, The human CRM1expressing rat cell lines were successfully established. 2, In the parental rat cells, Rex activity was at undetectable level, while in the human CRM1-transduced rat cells, the Rex activity was significantly enhanced to the level similar to the human cells. 3, Furthermore, the expression of human CRMI efficiently recovered the ability of HTLV-1 to produce viron proteins. 4, We could successfully produce a transgenic rat expressing human CRM1.

HIVとHTLV-1の高感受性小動物感染モデルを開発することは有用である。ラットでHIV感染はCD4とCCR5を発現させてやれば一定の増殖がある。又、HTLV-1においては不完全ではあるが感染モデルとして既に使用されている。しかし、ラット細胞でのHIV/HTLV-1の増殖は非常に効率が悪い。我々は、ラットrCRM1が効率のよいコファクターとならないためにウイルスのmRNA輸送因子Rev/Rexが働けないことを明らかにした。また、ヒトのhCRM1をラット細胞に導入するとRev/Rexの活性が回復することを示した。そこで、本研究目的は、ヒトhCRM1遺伝子を導入することによりHIV/HTLV-1感染トランスジェニックラットモデルの確立を試みることである。 今年度は先ず細胞レベルでヒトCRM1導入の効果を調べた。レトロベクターを用いてヒトCRM1を発現するラット細胞を作成し、特に、(1)内在するラットCRM1の優勢阻害効果の有無。(2)ヒトCRM1導入による細胞増殖への影響について検討した。その結果、(1)ヒト細胞に準じる量のHIV/HTLV-1のウイルス様粒子が生産されるようになることを見いだした。このことは、ラットCRM1の優勢阻害効果は無視しうる事を示している。また、(2)増殖への影響がある細胞と無い細胞があることが分かった。このことはヒトCRM1導入と同時にラットCRM1の発現を抑制することがトランスジェニックラットの作成に必要であることを示唆している。