Adjusting Heterodimeric Coiled-Coils (K/E Zipper) to Connect Autophagy-Inducing Peptide with Cell-Penetrating Peptide.

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.

Distinctive High Expression of Antiretroviral APOBEC3 Protein in Mouse Germinal Center B Cells.

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.

DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus.

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.

Deaminase-Independent Mode of Antiretroviral Action in Human and Mouse APOBEC3 Proteins.

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.

Intracellular delivery of a peptide nucleic acid-based hybrid of an autophagy inducing peptide with a cell-penetrating peptide.

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.

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing.

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.

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus.

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.

Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance.

CD8+ tissue-resident memory T cells (TRM cells) reside permanently in nonlymphoid tissues and provide a first line of protection against invading pathogens. However, the precise localization of CD8+ TRM 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+ TRM cells localize predominantly in specific niches created at the site of regeneration after tissue injury, whereas peripheral tissue-circulating CD8+ effector memory T cells (TEM 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 TRM niches. Furthermore, despite rigid segregation of TEM cells from the TRM niche, prime-pull strategy with cognate antigen enabled the conversion from TEM cells to TRM cells by creating de novo TRM niches. Such damage site-specific localization of CD8+ TRM cells may be important for efficient protection against secondary infections by respiratory pathogens.

Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest.

BACKGROUND: HIV-1 Vpr-mediated G2 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 G2 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 G2 arrest.

Two genetic determinants acquired late in Mus evolution regulate the inclusion of exon 5, which alters mouse APOBEC3 translation efficiency.

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 (Δ5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 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.

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells.

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.

Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase.

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.

HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1.

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.

Enhanced replication of human T-cell leukemia virus type 1 in T cells from transgenic rats expressing human CRM1 that is regulated in a natural manner.

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.

Reversed functional organization of mouse and human APOBEC3 cytidine deaminase domains.

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 Zn(2+) 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.

CRM1, an RNA transporter, is a major species-specific restriction factor of human T cell leukemia virus type 1 (HTLV-1) in rat cells.

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-1 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.

A multifunctional domain in human CRM1 (exportin 1) mediates RanBP3 binding and multimerization of human T-cell leukemia virus type 1 Rex protein.

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.

The carboxy-terminal region of the human immunodeficiency virus type 1 protein Rev has multiple roles in mediating CRM1-related Rev functions.

The human immunodeficiency virus type 1 (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.