Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays pivotal roles in both adaptive and innate immunities. GILT exhibits constitutive expression within antigen-presenting cells, whereas in other cell types, its expression is induced by interferon gamma (IFN-γ). Gaining insights into the precise molecular mechanism governing the induction of GILT protein by IFN-γ is of paramount importance for adaptive and innate immunities. In this study, we found that the 5' segment of GILT mRNA inhibited GILT protein expression regardless of the presence of IFN-γ. Conversely, the 3' segment of GILT mRNA suppressed GILT protein expression in the absence of IFN-γ, but it loses this inhibitory effect in its presence. Although the mTOR inhibitor rapamycin suppressed the induction of GILT protein expression by IFN-γ, the expression from luciferase sequence containing the 3' segment of GILT mRNA was resistant to rapamycin in the presence of IFN-γ, but not in its absence. Collectively, this study elucidates the mechanism behind GILT induction by IFN-γ: in the absence of IFN-γ, GILT mRNA is constitutively transcribed, but the translation process is hindered by both the 5' and 3' segments. Upon exposure to IFN-γ, a translation inhibitor bound to the 3' segment is liberated, and a translation activator interacts with the 3' segment to trigger the initiation of GILT translation.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.

Safety and efficacy of 5-aminolevulinic acid phosphate/iron in mild-to-moderate coronavirus disease 2019: A randomized exploratory phase II trial.

BACKGROUND: 5-aminolevulinic acid (5-ALA), a natural amino acid that is marketed alongside sodium ferrous citrate (SFC) as a functional food, blocks severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proliferation in vitro and exerts anti-inflammatory effects. In this phase II open-label, prospective, parallel-group, randomized trial, we aimed to evaluate the safety and efficacy of 5-ALA in patients with mild-to-moderate coronavirus disease 2019. METHODS: This trial was conducted in patients receiving 5-ALA/SFC (250/145 mg) orally thrice daily for 7 days, followed by 5-ALA/SFC (150/87 mg) orally thrice daily for 7 days. The primary endpoints were changes in SARS-CoV-2 viral load, clinical symptom scores, and 5-ALA/SFC safety (adverse events [AE] and changes in laboratory values and vital signs). RESULTS: A total of 50 patients were enrolled from 8 institutions in Japan. The change in SARS-CoV-2 viral load from baseline was not significantly different between the 5-ALA/SFC (n = 24) and control (n = 26) groups. The duration to improvement was shorter in the 5-ALA/SFC group than in the control group, although the difference was not significant. The 5-ALA/SFC group exhibited faster improvement rates in "taste abnormality," "cough," "lethargy," and "no appetite" than the control group. Eight AEs were observed in the 5-ALA/SFC group, with 22.7% of patients experiencing gastrointestinal symptoms (decreased appetite, constipation, and vomiting). AEs occurred with 750/435 mg/day in 25.0% of patients in the first phase and with 450/261 mg/day of 5-ALA/SFC in 6.3% of patients in the second phase. CONCLUSION: 5-ALA/SFC improved some symptoms but did not influence the SARS-CoV-2 viral load or clinical symptom scores over 14 days. The safety of 5-ALA/SFC in this study was acceptable. Further evaluation using a larger sample size or modified method is warranted.

Evaluation of a Triage Checklist for Mild COVID-19 Outpatients in Predicting Subsequent Emergency Department Visits and Hospitalization during the Isolation Period: A Single-Center Retrospective Study.

Managing mild illness in COVID-19 and predicting progression to severe disease are concerning issues. Here, we investigated the outcomes of Japanese patients with mild COVID-19, and identified triage risk factors for further hospitalization and emergency department (ED) visits at a single tertiary hospital. A triage checklist with 30 factors was used. Patients recommended for isolation were followed up for 10 days for subsequent ED visits or hospital admission. Overall, 338 patients (median age, 44.0; 45% women) visited the clinic 5.0 days (median) after symptom onset. Thirty-six patients were immediately hospitalized following triage; others were isolated. In total, 72 non-hospitalized patients visited the ED during their isolation, and 30 were hospitalized after evaluation for oxygen desaturation. The median ED visit and hospitalization durations after symptom onset were 5.0 and 8.0 days, respectively. The checklist factors associated with hospitalization during isolation were age > 50 years, body mass index > 25 kg/m2, hypertension, tachycardia with pulse rate > 100/min or blood pressure > 135 mmHg at triage, and >3-day delay in hospital visit after symptom onset. No patients died. Altogether, 80% of patients with mild COVID-19 could be safely isolated at home. Age, BMI, underlying hypertension, date after symptom onset, tachycardia, and systolic blood pressure at triage might be related to later hospitalization.

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

Unique Mode of Antiviral Action of a Marine Alkaloid against Ebola Virus and SARS-CoV-2.

Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.

Rab3a, a small GTP-binding protein, is required for the stabilization of the murine leukaemia virus Gag protein.

We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Efficient viral delivery of Cas9 into human safe harbor.

Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.

Cathepsin B Protease Facilitates Chikungunya Virus Envelope Protein-Mediated Infection via Endocytosis or Macropinocytosis.

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.

Human T-cell lymphotropic virus type-1 infection associated with sarcopenia: community-based cross-sectional study in Goto, Japan.

Sarcopenia is characterized by a progressive skeletal muscle disorder that involves the loss of muscle mass and low muscle strength, which contributes to increased adverse outcomes. Few studies have investigated the association between chronic infection and sarcopenia. This study aimed to examine the association between human T-cell lymphotropic virus type-1 (HTLV-1) and sarcopenia. We conducted a cross-sectional study and enrolled 2,811 participants aged ≥ 40 years from a prospective cohort study in Japanese community dwellers during 2017-2019. Sarcopenia was defined as low appendicular skeletal muscle mass and low handgrip strength. The association between HTLV-1 seropositivity and sarcopenia was assessed using multivariable logistic regression. Odds ratio (OR) and 95% confidence interval (CI) of sarcopenia were analysed using HTLV-1 seropositivity. We adjusted for age, sex, body mass index, physical activity, systolic blood pressure, glycated haemoglobin, low-density lipoprotein cholesterol, and smoking and drinking status. Of 2,811 participants, 484 (17.2%) HTLV-1 infected participants were detected. HTLV-1 infection was significantly associated with sarcopenia (adjusted OR 1.46, 95% CI 1.03-2.07, P = 0.034). HTLV-1 was associated with sarcopenia among community-dwelling adults. Active surveillance and early detection of asymptomatic HTLV-1 infection might be beneficial to reinforce countermeasures to inhibit the progress of HTLV infection-associated sarcopenia.

Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing.

Human immunodeficiency virus type 1 (HIV-1)-based viral vector is widely used as a biomaterial to transfer a gene of interest into target cells in many biological study fields including gene therapy. Vesicular stomatitis virus glycoprotein (VSV-G)-containing HIV-1 vector much more efficiently transduces various mammalian cells than other viral envelope proteins-containing vectors. Understanding the mechanism would contribute to development of a novel method of efficient HIV-1 vector production. HIV-1 vector is generally constructed by transient transfection of human 293T or African green monkey COS7 cells. It was found in this study that HIV-1 Gag protein is constitutively digested in lysosomes of African green monkey cells. Surprisingly, VSV-G elevated HIV-1 Gag protein levels, suggesting that VSV-G protects Gag protein from the lysosomal degradation. Unphosphorylated ezrin, but not phosphorylated ezrin, was detected in COS7 cells, and ezrin silencing elevated Gag protein levels in the presence of VSV-G. Expression of unphosphorylated ezrin reduced Gag protein amounts. These results indicate that unphosphorylated ezrin proteins inhibit the VSV-G-mediated stabilization of HIV-1 Gag protein. Trafficking of HIV-1 Gag-associated intracellular vesicles may be controlled by ezrin. Finally, this study found that ezrin silencing yields higher amount of VSV-G-pseudotyped HIV-1 vector.

The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

Cytoplasmic R-peptide of murine leukemia virus envelope protein negatively regulates its interaction with the cell surface receptor.

Cytoplasmic tails of envelope (Env) glycoproteins of many retroviruses inhibit their membrane fusion activity. The cytoplasmic 16-amino acid peptide of ecotropic murine leukemia virus (E-MLV) Env protein, called the R-peptide, also inhibits the membrane fusion activity of the Env protein. However, the molecular mechanism of the inhibition has not been elucidated yet. In this study, we found that R-peptide-containing Env protein of E-MLV binds to the cell surface receptor cationic amino acid transporter-1 (CAT-1) with weaker affinity than R-peptide-truncated Env protein. Consistent with this result, R-peptide-containing Env protein had less efficient inhibition of E-MLV vector infection than R-peptide-truncated Env protein. R-peptide truncation has been reported to induce conformational change in the surface subunit of E-MLV Env protein that interacts with the receptor. Taken together, our findings indicate that R-peptide truncation induces conformational change in the receptor-binding domain of the E-MLV Env protein and facilitates the Env-receptor interaction.

Role of Ezrin Phosphorylation in HIV-1 Replication.

Host-cell expression of the ezrin protein is required for CXCR4 (X4)-tropic HIV-1 infection. Ezrin function is regulated by phosphorylation at threonine-567. This study investigates the role of ezrin phosphorylation in HIV-1 infection and virion release. We analyzed the effects of ezrin mutations involving substitution of threonine-567 by alanine (EZ-TA), a constitutively inactive mutant, or by aspartic acid (EZ-TD), which mimics phosphorylated threonine. We also investigated the effects of ezrin silencing on HIV-1 virion release using a specific siRNA. We observed that X4-tropic HIV-1 vector infection was inhibited by expression of the EZ-TA mutant but increased by expression of the EZ-TD mutant, suggesting that ezrin phosphorylation in target cells is required for efficient HIV-1 entry. Expression of a dominant-negative mutant of ezrin (EZ-N) and ezrin silencing in HIV-1 vector-producing cells significantly reduced the infectivity of released virions without affecting virion production. This result indicates that endogenous ezrin expression is required for virion infectivity. The EZ-TD but not the EZ-TA inhibited virion release from HIV-1 vector-producing cells. Taken together, these findings suggest that ezrin phosphorylation in target cells is required for efficient HIV-1 entry but inhibits virion release from HIV-1 vector-producing cells.

Enterokinase Enhances Influenza A Virus Infection by Activating Trypsinogen in Human Cell Lines.

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines.

MicroRNA-3662 expression correlates with antiviral drug resistance in adult T-cell leukemia/lymphoma cells.

Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2 kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL.

Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles.

CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1), but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles.

Gamma-interferon-inducible, lysosome/endosome-localized thiolreductase, GILT, has anti-retroviral activity and its expression is counteracted by HIV-1.

The mechanism by which type II interferon (IFN) inhibits virus replications remains to be identified. Murine leukemia virus (MLV) replication was significantly restricted by γ-IFN, but not human immunodeficiency virus type 1 (HIV-1) replication. Because MLV enters host cells via endosomes, we speculated that certain cellular factors among γ-IFN-induced, endosome-localized proteins inhibit MLV replication. We found that γ-IFN-inducible lysosomal thiolreductase (GILT) significantly restricts HIV-1 replication as well as MLV replication by its thiolreductase activity. GILT silencing enhanced replication-defective HIV-1 vector infection and virion production in γ-IFN-treated cells, although γ-IFN did not inhibit HIV-1 replication. This result showed that GILT is required for the anti-viral activity of γ-IFN. Interestingly, GILT protein level was increased by γ-IFN in uninfected cells and env-deleted HIV-1-infected cells, but not in full-length HIV-1-infected cells. γ-IFN-induced transcription from the γ-IFN-activation sequence was attenuated by the HIV-1 Env protein. These results suggested that the γ-IFN cannot restrict HIV-1 replication due to the inhibition of γ-IFN signaling by HIV-1 Env. Finally, we found that 4,4'-dithiodipyridine (4-PDS), which inhibits S-S bond formation at acidic pH, significantly suppresses HIV-1 vector infection and virion production, like GILT. In conclusion, this study showed that GILT functions as a host restriction factor against the retroviruses, and a GILT mimic, 4-PDS, is the leading compound for the development of novel concept of anti-viral agents.