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IgA is the most abundant isotype of antibodies and provides a first line of defense at the mucosa against pathogens invading the host. It has been widely accepted that the mucosal IgA response provided by vaccination requires mucosal inoculation, and intranasal inoculation has been proposed for vaccines against influenza virus. Considering the difficulty of intranasal vaccination in infants or elderly people, however, parenteral vaccination that provides the mucosal IgA response is desirable. Here, we demonstrate that subcutaneous immunization with zymosan, a yeast cell wall constituent known to be recognized by Dectin-1 and TLR2, potentiates the production of antigen-specific IgA antibodies in the sera and airway mucosa upon intranasal antigen challenge. We confirmed that the antigen-specific IgA-secreting cells accumulated in the lung and nasal-associated lymphoid tissues after the antigen challenge. Such an adjuvant effect of zymosan in the primary immunization for the IgA response depended on Dectin-1 signaling, but not on TLR2. The IgA response to the antigen challenge required both antigen-specific memory B and T cells, and the generation of memory T cells, but not memory B cells, depended on zymosan as an adjuvant. Finally, we demonstrated that subcutaneous inoculation of inactivated influenza virus with zymosan, but not with alum, mostly protected the mice from infection with a lethal dose of a heterologous virus strain. These data suggest that zymosan is a possible adjuvant for parenteral immunization that generates memory IgA responses to respiratory viruses such as influenza virus.

Influenza viruses are a major public health problem. Vaccines are the best available countermeasure to induce effective immunity against infection with seasonal influenza viruses; however, the breadth of antibody responses in infection versus vaccination is quite different. Here, we show that nasal infection controls two sequential processes to induce neutralizing IgG antibodies recognizing the hemagglutinin (HA) of heterotypic strains. The first is viral replication in the lung, which facilitates exposure of shared epitopes that are otherwise hidden from the immune system. The second process is the germinal center (GC) response, in particular, IL-4 derived from follicular helper T cells has an essential role in the expansion of rare GC-B cells recognizing the shared epitopes. Therefore, the combination of exposure of the shared epitopes and efficient proliferation of GC-B cells is critical for generating broadly-protective antibodies. These observations provide insight into mechanisms promoting broad protection from virus infection., The reasons why influenza infection promotes a broader antibody response compared with vaccines are not fully understood. Here the authors show that unmasking of haemagglutinin epitopes and IL-4 signals in the germinal centre contribute to broader antibody responses after infection.

Influenza viruses are a major public health problem, causing severe respiratory diseases. Vaccines offer the effective protective strategy against influenza virus infection. However, the systemic and adaptive immune responses to infection and vaccination are quite different. Inactivated vaccines are the best available countermeasure to induce effective antibodies against the emerged virus, but the response is narrow compared with potential breadth of virus infection. There is solid evidence to indicate that antibody responses to natural infection are relatively broad and exhibit quite different immunodominance patterns. Furthermore, T follicular helper cells (TFH) and germinal center (GC) responses play a central role in generating broad protective antibodies. In this review, we discuss recent advances on the contribution of TFH and GC responses to the breadth of antibody responses.

Tissue-resident lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells, and lung airway TRM cells are primarily maintained by the continuous seeding of cells from the lung interstitium., Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance., Graphical Abstract

The lung is an important line of defense that is exposed to respiratory infectious pathogens, including viruses. Lung epithelial cells and/or alveolar macrophages are initially targeted by respiratory viruses. Once respiratory viruses invade the cells of the lung, innate immunity is activated to inhibit viral replication. Innate immune signaling also activates virus-specific adaptive immune responses. The helper T cells play pivotal roles in the humoral and cellular adaptive immune responses. Helper T cells are categorized into several distinct subsets (e.g., TH1, TH2, TFH, TH17, and Treg), differentiated by their corresponding signature cytokine production profiles. Helper T cells migrate into the airways and the lung after respiratory virus infections. The behavior of the helper T cells differs with each respiratory virus-in some cases, the response is beneficial; in other cases, it is harmful. Here, the general mechanisms underlying helper T cell responses to viral infections are summarized, and functions and reactions of the helper T cells against some respiratory viral infections are discussed. In influenza virus infections, TH1 cells, which regulate the cytotoxic T lymphocytes and IgG2 responses, are efficiently activated. TFH cells required for highly specific and memory humoral responses are also activated on influenza infections. In infections with respiratory syncytial virus and rhinovirus, TH2 cells develop in the lung and contribute to pathogenesis. In many cases, Treg cells inhibit excessive virus-specific T cell responses that can contribute to viral pathogenicity.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memoryCD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM)and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that,following a critical number of cell divisions, memory precursors down regulate CCR7 and upregulate IL-2Rb, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM. Refereed/Peer-reviewed

Specific deletion of suppressor of cytokine signaling 3 (Socs3) in keratinocytes can cause severe skin inflammation with infiltration of immune cells. The molecular mechanisms and key regulatory pathways involved in these processes remain elusive. To investigate the role of Socs3 in keratinocytes, we generated and analyzed global RNA-Seq profiles from Socs3 conditional knockout (cKO) mice of two different ages (2 and 10 weeks). Over 400 genes were significantly regulated at both time points. Samples from 2-week-old mice exhibited down-regulation of genes involved in keratin-related functions and up-regulation of genes involved in lipid metabolism. At week 10, multiple chemokine and cytokine genes were up-regulated. Functional annotation revealed that the genes differentially expressed in the 2-week-old mice play roles in keratinization, keratinocyte differentiation, and epidermal cell differentiation. By contrast, differentially expressed genes in the 10-week-old animals are involved in acute immune-related functions. A group of activator protein-1–related genes were highly up-regulated in Socs3 cKO mice of both ages. This observation was validated using qRT-PCR by SOCS3-depleted human keratinocyte–derived HaCaT cells. Our results suggest that, in addition to participating in immune-mediated pathways, SOCS3 also plays important roles in skin barrier homeostasis.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

The conformational dynamics of the HIV-1 envelope glycoprotein gp120 and gp41 (Env) remains poorly understood. Here we examined how the V3 loop conformation is regulated in the liganded state using a panel of recombinant HIV-1NL4-3 clones bearing HIV-1AD8 Env by two experimental approaches, one adopting a monoclonal neutralizing antibody KD-247 (suvizumab) that recognizes the tip of the V3 loop, and the other assessing the function of the V3 loop. A significant positive correlation of the Env-KD-247 binding was detected between the liganded and unliganded conditions. Namely, the mutation D163G located in the V2 loop, which enhances viral susceptibility to KD-247 by 59.4-fold, had little effect on the sCD4-induced increment of the virus-KD-247 binding. By contrast, a virus with the S370N mutation in the C3 region increased the virus-KD-247 binding by 91.4-fold, although it did not influence the KD-247-mediated neutralization. Co-receptor usage and the susceptibility to CCR5 inhibitor Maraviroc were unaffected by D163G and S370N mutations. Collectively, these data suggest that the conformation of the liganded V3-loop of HIV-1AD8 Env is still under regulation of other Env domains aside from the V3 loop, including V2 and C3. Our results give an insight into the structural properties of HIV-1 Env and viral resistance to entry inhibitors by non-V3 loop mutations.

It has long been believed that the generation of high-affinity neutralizing antibodies is critical for antiviral humoral immunity. The production of high-affinity antibodies depends on the formation of germinal centers (GC) in lymph nodes and a subset of T cells called follicular helper T (TFH) cells in GCs. However, we discovered that T helper 1 (TH1) cells induce low-affinity IgG2 antibodies sufficient for preventing influenza A virus (IAV) infection.1

A novel HIV-1 inhibitor, 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (compound 1), was identified from a compound library screened for the ability to inhibit HIV-1 replication. EC(50) values of compound 1 were found to range from 107.9 to 145.4 nm against primary HIV-1 clinical isolates. In in vitro assays, HIV-1 reverse transcriptase (RT) activity was inhibited by compound 1 with an EC(50) of 4.3 μm. An assay for resistance to compound 1 selected a variant of HIV-1 with a RT mutation (RT(L100I)); this frequently identified mutation confers mild resistance to non-nucleoside RT inhibitors (NNRTIs). A recombinant HIV-1 bearing RT(L100I) exhibited a 41-fold greater resistance to compound 1 than the wild-type virus. Compound 1 was also effective against HIV-1 with RT(K103N), one of the major mutations that confers substantial resistance to NNRTIs. Computer-assisted docking simulations indicated that compound 1 binds to the RT NNRTI binding pocket in a manner similar to that of efavirenz; however, the putative compound 1 binding site is located further from RT(K103) than that of efavirenz. Compound 1 is a novel NNRTI with a unique drug-resistance profile.

The skin barrier acts as the first line of defense against constant exposure to biological, microbial, physical, and chemical environmental stressors. Dynamic interplay between defects in the skin barrier, dysfunctional immune responses, and environmental stressors are major factors in the development of atopic dermatitis (AD). A systems biology modeling approach can yield significant insights into these complex and dynamic processes through integration of prior biological data.We sought to develop a multiscale mathematical model of AD pathogenesis that describes the dynamic interplay between the skin barrier, environmental stress, and immune dysregulation and use it to achieve a coherent mechanistic understanding of the onset, progression, and prevention of AD.We mathematically investigated synergistic effects of known genetic and environmental risk factors on the dynamic onset and progression of the AD phenotype, from a mostly asymptomatic mild phenotype to a severe treatment-resistant form.Our model analysis identified a "double switch," with 2 concatenated bistable switches, as a key network motif that dictates AD pathogenesis: the first switch is responsible for the reversible onset of inflammation, and the second switch is triggered by long-lasting or frequent activation of the first switch, causing irreversible onset of systemic TOur mathematical analysis of the bistable switch predicts that genetic risk factors decrease the threshold of environmental stressors to trigger systemic T

Cyclin T1 (CCNT1), a gene containing nine exons, forms the positive transcription elongation factor b (P-TEFb) complex and regulates a wide variety of biological processes including transcription. We discovered a novel splice variant of CCNT1 that lacks exon 7 (dE7). RT-PCR analysis revealed that the dE7 transcript was detected in almost all tissues examined. The dE7/FL transcript ratio was high in quiescent peripheral blood mononuclear cells (PBMC) and in tissues poor in cell division; however, it was low in activated PBMC and in tissues with high cell proliferative potential. These results suggest that exon 7 skipping is linked to cell cycle progression. Increasing the dE7/FL transcript ratio resulted in the reduction of CCNT1 protein levels, indicating that the expression of CCNT1 protein is controlled by exon skipping. Exon 7 skipping yields a +1 frameshift at exon 8, which generates a premature termination codon (PTC). The dE7 transcript levels increased when cells were treated with the protein synthesis inhibitor cycloheximide (CHX) or a kinase inhibitor wortmannin (WORT), whilst the FL transcript levels were unchanged, suggesting that the dE7 transcript is a target of nonsense-mediated decay (NMD). Importantly, reduction of dE7 transcript by WORT correlated well with the decrement of CCNT1 protein expression. The dE7 transcript would produce an approximately 23kDa protein that covers approximately 70% of the cyclin box. The ectopically expressed dE7 protein physically interacted with CDK9 and competed with FL CCNT1 for CDK9, thus should act dominant-negatively on FL CCNT1. The replication of human immunodeficiency virus type 1 (HIV-1), heavily dependent on the CCNT1 function, was inhibited by dE7 protein through the attenuation of Tat/long terminal repeat (LTR)-driven transcription. Taken together, these results suggest that dE7 is a novel splice variant that regulates the expression and function of CCNT1.

An artificial antigen forming the C34 trimeric structure targeting membrane-fusion mechanism of HIV-1 has been evaluated as an HIV vaccine. The C34 trimeric molecule was previously designed and synthesized using a novel template with C3-symmetric linkers by us. The antiserum produced by immunization of the C34 trimeric form antigen showed 23-fold higher binding affinity for the C34 trimer than for the C34 monomer and showed significant neutralizing activity. The present results suggest effective strategies of the design of HIV vaccines and anti-HIV agents based on the native structure mimic of proteins targeting dynamic supramolecular mechanisms in HIV fusion.

A humanized neutralizing antibody, KD-247, targets the V3 loop of HIV-1 Env. HIV-1 bearing the GPGR sequence at the V3 loop is potentially susceptible to KD-247. However, not all GPGR-positive HIV-1 isolates are neutralized by KD-247. We examined the potential mechanism by which the susceptibility of HIV-1 to KD-247-mediated neutralization is regulated.We searched for nonepitope neutralization regulatory (NNR) mutations that sensitize GPGR-bearing HIV-1AD8 to KD-247 and mapped the locations of such mutations relative to the V3 loop.: We generated a functional HIV-1AD8 Env library, and evaluated the viral susceptibility to KD-247 by measuring the half-inhibitory concentration (IC50) to KD-247 on TZM-bl cell assay.We identified nine KD-247-sensitizing NNR mutations from 30 mutations in various regions of gp120, including the V1/V2 loop, C2, V3 loop, C4, and C5. They specifically affected KD-247-mediated neutralization, as they did not affect the b12-mediated neutralization. When combined, the KD-247-sensitizing NNR mutations additively sensitized the virus to KD-247 by up to 10 000 folds. The KD-247-sensitizing NNR mutations increased KD-247 binding to the virion. Notably, the NNR mutation in C4 coincides with the CD4-binding site of gp120.Given that most of the KD-247-sensitizing NNR mutations are remote from V3 loop, it is reasonable to hypothesize that the steady-state, local conformation of the V3 loop is regulated by the interdomain contact of gp120. Our mutational analysis complements crystallographic studies by helping provide a better understanding of the steady-state conformation and the functional geometry of Env.

Human immunodeficiency virus (HIV) Gag protein targets to the plasma membrane and assembles into viral particles. In the next round of infection, the mature Gag capsids disassemble during viral entry. Thus, Gag plays a central role in the HIV life cycle. Using a yeast membrane-associated two-hybrid assay based on the SOS-RAS signaling system, we developed a system to measure the Gag-Gag interaction and isolated 6 candidates for Gag assembly inhibitors from a chemical library composed of 20,000 small molecules. When tested in the human MT-4 cell line and primary peripheral blood mononuclear cells, one of the candidates, 2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine (BMMP), displayed an inhibitory effect on HIV replication, although a considerably high dose was required. Unexpectedly, neither particle production nor maturation was inhibited by BMMP. Confocal microscopy confirmed that BMMP did not block Gag plasma membrane targeting. Single-round infection assays with envelope-pseudotyped and luciferase-expressing viruses revealed that BMMP inhibited HIV replication postentry but not simian immunodeficiency virus (SIV) or murine leukemia virus infection. Studies with HIV/SIV Gag chimeras indicated that the Gag capsid (CA) domain was responsible for the BMMP-mediated HIV postentry block. In vitro studies indicated that BMMP accelerated disassembly of HIV cores and, conversely, inhibited assembly of purified CA protein in a dose-dependent manner. Collectively, our data suggest that BMMP primarily targets the HIV CA domain and disrupts viral infection postentry, possibly through inducing premature disassembly of HIV cores. We suggest that BMMP is a potential lead compound to develop antiretroviral drugs bearing novel mechanisms of action.

A simple, efficient and reproducible method to transduce proteins into mammalian cells has not been established. Here we describe a novel protein transduction method based on a lentiviral vector. We have developed a method to package several thousand foreign protein molecules into a lentivirus-like nanoparticle (LENA) and deliver them into mammalian cells. In this proof-of-concept study, we used β-lactamase (BlaM) as a reporter molecule. The amino-terminus of BlaM was fused to the myristoylation signal of lyn, which was placed upstream of the amino-terminus of Gag (BlaM-gag-pol). By co-transfection of plasmids encoding BlaM-gag-pol and vesicular stomatitis virus-G (VSV-G) into 293T cells, LENA were produced containing BlaM enzyme molecules as many as Gag per capsid, which has been reported to be ∼5000 molecules, but lacking the viral genome. Infection of 293T and MT-4 cells by VSV-G-pseudotyped BlaM-containing LENA led to successful transduction of BlaM molecules into the cell cytoplasm, as detected by cleavage of the fluorescent BlaM substrate CCF2-AM. LENA-mediated transient protein transduction does not damage cellular DNA, and the preparation of highly purified protein is not necessary. This technology is potentially useful in various basic and clinical applications.

Anti-HIV peptides with inhibitory activity against HIV-1 integrase (IN) have been found in overlapping peptide libraries derived from HIV-1 gene products. In a strand transfer assay using IN, inhibitory active peptides with certain sequential motifs related to Vpr- and Env-derived peptides were found. The addition of an octa-arginyl group to the inhibitory peptides caused a remarkable inhibition of the strand transfer and 3'-end-processing reactions catalyzed by IN and significant inhibition against HIV replication.

To help understand the dynamic nature of membrane fusion induced by the human immunodeficiency virus-1 (HIV-1) envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for Renilla luciferase achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains were replaced with that of glycophorin A or vesicular stomatitis virus G-protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.

SummaryEnveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here we used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. We also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glycoprotein- and dynamin-dependent fusion with intracellular compartments.

The fusion between viral and cellular membranes is the first critical step of the enveloped viral infection. This is promoted by the drastic conformational change of the viral fusion protein. The conformational change is driven by various cues that are different in each fusion protein. The divergent nature of the induction mechanism of fusion proteins tells us that the regulation of membrane fusion process is substantially important to viral infection. Historically, enveloped viruses were categorized into pH-dependent and pH-independent groups for their entry processes. It has been thought that the pH-independent viruses mainly fuse to cell membrane at the cell surface whereas pH-dependent viruses fuse to endosomal membrane. However, the recent studies suggest that some pH-independent viruses including Human Immunodeficiency Virus (HIV) also utilize the endocytosis pathway to achieve infection. In addition, it has been revealed that the host factors other than receptors play crucial roles in the entry of enveloped viruses. This review summarizes the entry process of enveloped viruses and focuses on the current topics of HIV entry.

Objective Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. Methods HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. Results HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. Conclusion These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.

Takeuchi et al. demonstrate that CRTAM identifies CD4 T cells with cytotoxic function, and present new insights into CD4+CTL development., Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene.

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.

The toll-like receptor (TLR)-7 has been shown to sense the retroviral infection. However, a surrogate sensor has been implicated. We examined whether retrovirus serves as a TLR3 ligand in human cells by utilizing cell lines LNCaP and PC-3 lacking TLR7, and the xenotropic murine leukemia virus-relamoted virus (XMRV) insensitive to human tripartite motif-containing (TRIM) 5, a newly characterized pattern recognition receptor (PRR). A dominant-negative TLR3 or a chemical inhibitor of TLR3 attenuated the XMRV-induced IP-10/CXCL10 expression, a marker of TLR3 response. These data clearly indicated that retroviral infection exemplified by XMRV activates the TLR3 signal in human cells.

Development of a therapeutic application of CASP3/caspase 3/CPP32, an executor of apoptosis, has been challenging because regulation of its activation is complicated. This study aimed to inhibit cancer cell growth and human immunodeficiency virus type 1 (HIV-1) propagation through a CASP3 mutant, CASP3*, activable by HIV-1-encoded aspartate protease. Active CASP3* was delivered to leukemic cells using a protein transduction vehicle, the lentivirus-like nanoparticle (LENA), which should contain thousands of CASP3*-Gag protein molecules and release the activated CASP3* into the target cell cytoplasm. CASP3*-LENA induced apoptosis in various types of leukemic cells. In addition to being effective against leukemic cells, constitutive expression of CASP3* restricted HIV-1 propagation in SUP-T1 cells. The attenuation of HIV-1 replication in SUP-T1/CASP3* cells was attributed to the elimination of HIV-1-infected cells by apoptosis. These data suggest that CASP3* has therapeutic potential against both lymphoid malignancies and HIV-1 infection.

Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

Rho GTPases are able to influence the replication of human immunodeficiency virus type 1 (HIV-1). However, little is known about the regulation of HIV-1 replication by guanine nucleotide dissociation inhibitors (GDIs), one of the three major regulators of the Rho GTPase activation cycle. From a T cell-based cDNA library screening, ARHGDIB/RhoGDIβ, a hematopoietic lineage-specific GDI family protein, was identified as a negative regulator of HIV-1 replication. Up-regulation of ARHGDIB attenuated the replication of HIV-1 in multiple T cell lines. The results showed that (1) a significant portion of RhoA and Rac1, but not Cdc42, exists in the GTP-bound active form under steady-state conditions, (2) ectopic ARHGDIB expression reduced the F-actin content and the active forms of both RhoA and Rac1, and (3) HIV-1 infection was attenuated by either ectopic expression of ARHGDIB or inhibition of the RhoA signal cascade at the HIV-1 Env-dependent early phase of the viral life cycle. This is in good agreement with the previous finding that RhoA and Rac1 promote HIV-1 entry by increasing the efficiency of receptor clustering and virus-cell membrane fusion. In conclusion, the ARHGDIB is a lymphoid-specific intrinsic negative regulator of HIV-1 replication that acts by simultaneously inhibiting RhoA and Rac1 functions.

Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solutions. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solutions of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alkaline buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alkaline buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.

Immunocompromised individuals, including those infected with human immunodeficiency virus (HIV), are at increased risk of Epstein-Barr virus (EBV)-associated aggressive B cell malignancies such as Burkitt's lymphoma (BL) or diffuse large B cell lymphoma (DLBCL). Differential diagnosis of these lymphomas requires histopathological, immunohistochemical and cytogenetic assessments. Rapid, less invasive approaches to the diagnosis of EBV-associated B cell lymphomas are needed. Here, high-throughput cytokine profiling of BL cell lines and EBV-transformed B lymphoblastoid cell lines (B-LCL), representing DLBCL, was carried out. By monitoring the production of 42 different cytokines, unique cytokine signatures were identified for BL and B-LCL/DLBCL. The BL cells produced interleukin (IL)-10, 10 kDa interferon gamma-induced protein (IP-10)/CXCL10, macrophage-derived chemokine (MDC)/CCL22, macrophage inflammatory protein (MIP)-1α/CCL3 and MIP-1β/CCL4. In addition to these five cytokines, the cytokine signature of B-LCL/DLBCL cells included IL-8/CXCL8, IL-13, platelet-derived growth factor (PDGF)-AA, and regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5. Epstein-Barr virus latency was responsible for the increased production of IL-10, MDC/CCL22 and MIP-1α/CCL3 in BL cells, suggesting that EBV-mediated BL-genesis involves these three cytokines. These results suggest that high-throughput cytokine profiling might be a valuable tool for the differential diagnosis and might deepen our understanding of the pathogenesis of EBV-associated B cell malignancies.

A simple and real-time cell-based assay of membrane fusion employing a pair of engineered novel reporter proteins is described. The reporter proteins are chimeras of split Renilla luciferase (RL) and split green fluorescent protein (GFP). This reporter allows us to perform both quantitative (RL mode) and visible (GFP mode) membrane fusion assays in live cells. The kinetic assay enabled by this method helps understand the mechanism of membrane fusion mediated by a viral envelope protein. This assay system is also suitable for the screening of potential inhibitors. The timing of inhibition by a particular inhibitor can be analyzed by time-dependent addition of the inhibitor. Although this unit demonstrates the application of the method for the analysis of HIV-1 envelope protein, the reporter can be applied to analyses of various other viral envelope proteins.

Avian Sarcoma and Leukosis Virus (ASLV) entry into cells proceeds through two major steps -priming by cognate receptors on the cell surface followed by low pH-induced fusion with endosomes. Subtype A viruses are able to utilize two naturally occurring isoforms of the TVA receptor for infection, the GPI-anchored (TVA800) and the transmembrane (TVA950) receptors, which are localized to different membrane domains. We monitored the consecutive steps of ASLV entry into cells expressing these receptor isoforms, including endocytosis, delivery into acidic compartments, and fusion with endosomes. Cells expressing TVA950 internalized ASLV much faster than those expressing the alternative receptor. In both cell lines, fusion occurred shortly after internalization, suggesting that virus uptake was the rate-limiting step of entry. We found that ASLV fusion with endosomes proceeded through two relatively long-lived intermediates, a hemifusion-like intermediate and a small fusion pore. The lifetime of these intermediates was dependant on TVA receptor isoforms and their expression levels. TVA950 supported more robust fusion intermediates compared to TVA800. These findings are consistent with ASLV trafficking by via TVA950 to endosomal compartments that are more conducive to fusion. Alternatively, transmembrane receptor might more efficiently prime the viral fusion proteins compared to the GPI-anchored receptor. This work has been supported by the NIH AI053668 grant.

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes., Author Summary Enveloped viruses are taken up into host cells after they bind to a specific receptor on the cell surface. These viruses are often delivered to specific compartments known as endosomes which have an acid environment that can trigger the virus and endosome membranes to fuse, allowing virus entry into the cell interior. In spite of extensive studies, key steps of this process are poorly understood. To examine the mechanism of virus-endosome fusion, we took advantage of a model retrovirus (ASLV). We imaged the entry of “labeled” ASLV into cells using fluorescence microscopy and found important differences between ASLV entry, depending upon whether the virus was attached to a transmembrane receptor or to a lipid-anchored receptor. A transmembrane receptor supported faster virus uptake followed by the formation of larger and more stable fusion pores, which was somewhat delayed relative to ASLV entry into acidic endosomes. This could be due, in part, to a relatively long-lived hemifusion intermediate that preceded the formation of a fusion pore in cells expressing a transmembrane, but not a lipid-anchored receptor. These findings shed light on functional consequences of virus entry via distinct endocytic routes and on critical fusion intermediates leading to infection.

Background The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. Results A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. Conclusions The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.

Recent work from our laboratory (Miyauchi et al. 2009, Cell 137:433-444) provided functional evidence for HIV-1 entry via an endocytic pathway. In contrast, HIV-1 fusion with the plasma membrane (PM) did not proceed beyond the lipid mixing step. We have therefore assessed the ability of other enveloped viruses to fuse with the PM of permissive cells. Imaging of single amphotropic Murine Leukemia Virus particles revealed complete endoplasmic fusion with occasional lipid mixing at the PM. Next, we tested whether low pH-dependent viruses can be forced to fuse with the PM by lowering the external pH. When retroviral cores pseudotyped with the Semliki Forest Virus E1E2 glycoproteins were bound to cells and exposed to acidic pH, lipid mixing, but not viral content transfer was observed. Likewise, Avian Sarcoma and Leukosis Virus (ASLV) that enters target cells via a receptor-mediated endocytosis followed by low pH-induced fusion with endosomes failed to release its content at the PM upon reducing the pH. These results demonstrate a surprisingly strong preference for endosomal fusion among pH-dependent and pH-independent viruses, suggesting the existence of a fusion block at the PM and/or the involvement of an endosomal factor(s) facilitating the viral fusion. Consistent with the latter mechanism, inhibition of the dynamin function blocked endosomal fusion of HIV-1, ASLV and several other viruses. Together, these results are consistent with the involvement of cellular factors in facilitating the viral entry via endosomes. Supported by NIH grants GM054787 and AI053668.

The HIV envelope (Env) glycoprotein mediates membrane fusion through sequential interactions with CD4 and coreceptors, followed by the refolding of the transmembrane gp41 subunit into the stable 6-helix bundle (6HB) conformation. Synthetic peptides derived from the gp41 C-terminal heptad repeat domain (C-peptides) potently inhibit fusion by binding to the gp41 pre-bundle intermediates and blocking their conversion into the 6HB. Our recent work revealed that HIV-1 enters cells by fusing with endosomes, but not with the plasma membrane. These studies also showed that, for the large part, gp41 pre-bundles progress toward 6HBs in endosomal compartments and are thus protected from external fusion inhibitors. Here, we examined the consequences of endocytic entry on the gp41 pre-bundle exposure and on the virus' sensitivity to C-peptides. The rates of CD4 and coreceptor binding, as well as the rate of productive receptor-mediated endocytosis, were measured by adding specific inhibitors of these steps at varied times of virus-cell incubation. Following the CD4 binding, CCR5-tropic viruses recruited a requisite number of coreceptors much faster than CXCR4-tropic viruses. The rate of subsequent uptake of ternary Env-CD4-coreceptor complexes did not correlate with the kinetics of coreceptor engagement. These measurements combined with kinetic analyses enabled the determination of the lifetime of pre-bundle intermediates on the cell surface. Overall, these lifetimes correlated with the inhibitory potency of C-peptides. On the other hand, the basal sensitivity to peptides varied considerably among diverse HIV-1 isolates and ranked similarly with their susceptibility to inactivation by soluble CD4. We conclude that both the longevity of gp41 intermediates and the extent of irreversible conformational changes in Env upon CD4 binding determine the antiviral potency of C-peptides., Author Summary The human immunodeficiency virus (HIV) envelope glycoprotein (Env) mediates fusion between the viral and cell membranes. The fusion is initiated by Env-receptor interactions and is followed by coreceptor binding and refolding of the transmembrane gp41 subunit. The gp41 refolding proceeds through several distinct intermediates, culminating in the formation of a final helical bundle structure which is blocked by inhibitory peptides targeting the complementary domains of gp41. We have recently shown that the exposure time of gp41 intermediates on the cell surface is limited by productive HIV endocytosis leading to fusion with endosomes. Here, we measured the rates of progression of different HIV isolates through distinct intermediate steps accessible to fusion inhibitors and correlated these rates with the inhibitory potency of peptides against these viruses. Whereas the potency of peptides was proportional to the lifetime of gp41 intermediates on the cell surface, the baseline sensitivity of the virus was also Env context-dependent. Higher concentrations of these inhibitors were required to block fusion induced by glycoproteins that were more resistant to inactivation by the soluble receptor. Collectively, these findings imply that both the kinetic factors and the stability of Env-receptor complexes control the HIV sensitivity to inhibitory peptides.

Viruses whose fusion proteins are activated by interactions with cellular receptors at neutral pH, including HIV-1, are assumed to fuse directly with the plasma membrane. However, direct virus fusion at the cell surface has not been explicitly demonstrated. To differentiate between HIV-1 fusion with the plasma membrane (PM) and with endosomal membrane (EM), we performed time-resolved single virus imaging. Pseudoviruses bearing HIV-1 Env glycoprotein were generated and co-labeled with a content marker (Gag-GFP) and a red lipophilic membrane dye. Upon virus maturation, the Gag-GFP is cleaved by viral protease yielding a smaller GFP-tagged fragment that is readily released from virions permeabilized with saponin. This marker thus provided a convenient means to detect small pore formation during virus-cell fusion. Double-labeled viruses were adhered in the cold to HeLa-derived cells expressing CD4 and coreceptors. Fusion was triggered by shifting to 37°C and monitored by laser scanning confocal microscopy. Imaging of single HIV-cell fusion revealed the occurrence of both PM and EM fusion events. Fluorescent viruses undergoing PM fusion transferred their lipid marker (hemifusion), but not content marker (full fusion). By contrast, full fusion with an endosome was consistently observed. These EM fusion events were manifested in disappearance of a content marker, but not the lipid dye due to its limited dilution in an endosomal membrane. These results demonstrate that, contrary to a common perception, HIV-1 enters HeLa-derived target cells by receptor/coreceptor-mediated endocytosis followed by fusion with endosomes and delivery of viral nucleocapsid into the cytosol.

HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.

HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.

The C-terminal cytoplasmic domain of the metastatic potentiator CXCR4 regulates its function and spatiotemporal expression. However, little is known about the mechanism underlying constitutive internalization of CXCR4 compared to internalization mediated by its ligand, stromal cell-derived factor-1 alpha (SDF-1α)/CXCL12. We established a system to analyze the role of the CXCR4 cytoplasmic tail in steady-state internalization using the NP2 cell line, which lacks endogenous CXCR4 and SDF-1α. Deleting more than six amino acids from the C-terminus dramatically reduced constitutive internalization of CXCR4. Alanine substitution mutations revealed that three of those amino acids Ser344 Glu345 Ser346 are essential for efficient steady-state internalization of CXCR4. Mutating Glu345 to Asp did not disrupt internalization, suggesting that the steady-state internalization motif is S(E/D)S. When responses to SDF-1α were tested, cells expressing CXCR4 mutants lacking the C-terminal 10, 14, 22, 31 or 44 amino acids did not show downregulation of cell surface CXCR4 or the cell migration induced by SDF-1α. Interestingly, however, we identified two mutants, one with E344A mutation and the other lacking the C-terminal 17 amino acids, that were defective in constitutive internalization but competent in ligand-promoted internalization and cell migration. These data demonstrate that ligand-dependent and -independent internalization is genetically separable and that, between amino acids 336 and 342, there is a negative regulatory element for ligand-promoted internalization. Potential involvement of this novel motif in cancer metastasis and other CXCR4-associated disorders such as warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome is discussed. (Cancer Sci 2007; 98: 373–379)