Flagellin Restricts HIV-1 Infection of Macrophages through Modulation of Viral Entry Receptors and CC Chemokines.

Both bacteria product flagellin and macrophages are implicated in HIV-1 infection/disease progression. However, the impact of their interaction on HIV-1 infection and the associated mechanisms remain to be determined. We thus examined the effect of the flagellins on HIV-1 infection of primary human macrophages. We observed that the pretreatment of macrophages with the flagellins from the different bacteria significantly inhibited HIV-1 infection. The mechanistic investigation showed that the flagellin treatment of macrophages downregulated the major HIV-1 entry receptors (CD4 and CCR5) and upregulated the CC chemokines (MIP-1α, MIP-1ß and RANTES), the ligands of CCR5. These effects of the flagellin could be compromised by a toll-like receptor 5 (TLR5) antagonist. Given the important role of flagellin as a vaccine adjuvant in TLR5 activation-mediated immune regulation and in HIV-1 infection of macrophages, future investigations are necessary to determine the in vivo impact of flagellin-TLR5 interaction on macrophage-mediated innate immunity against HIV-1 infection and the effectiveness of flagellin adjuvant-based vaccines studies.

KDM5A/B contribute to HIV-1 latent infection and survival of HIV-1 infected cells.

Combinational antiretroviral therapy (cART) suppresses human immunodeficiency virus type 1 (HIV-1) viral replication and pathogenesis in acquired immunodeficiency syndrome (AIDS) patients. However, HIV-1 remains in the latent stage of infection by suppressing viral transcription, which hinders an HIV-1 cure. One approach for an HIV-1 cure is the "shock and kill" strategy. The strategy focuses on reactivating latent HIV-1, inducing the viral cytopathic effect and facilitating the immune clearance for the elimination of latent HIV-1 reservoirs. Here, we reported that the H3K4 trimethylation (H3K4me3)-specific demethylase KDM5A/B play a role in suppressing HIV-1 Tat/LTR-mediated viral transcription in HIV-1 latent cells. Furthermore, we evaluated the potential of KDM5-specific inhibitor JQKD82 as an HIV-1 "shock and kill" agent. Our results showed that JQKD82 increases the H3K4me3 level at HIV-1 5' LTR promoter regions, HIV-1 reactivation, and the cytopathic effects in an HIV-1-latent T cell model. In addition, we identified that the combination of JQKD82 and AZD5582, a non-canonical NF-κB activator, generates a synergistic impact on inducing HIV-1 lytic reactivation and cell death in the T cell. The latency-reversing potency of the JQKD82 and AZD5582 pair was also confirmed in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 aviremic patients and in an HIV-1 latent monocyte. In latently infected microglia (HC69) of the brain, either deletion or inhibition of KDM5A/B results in a reversal of the HIV-1 latency. Overall, we concluded that KDM5A/B function as a host repressor of the HIV-1 lytic reactivation and thus promote the latency and the survival of HIV-1 infected reservoirs.

Cytosolic DNA sensor activation inhibits HIV infection of macrophages.

Cytosolic recognition of microbial DNA in macrophages results in the activation of the interferon (IFN)-dependent antiviral innate immunity. Here, we examined whether activating DNA sensors in peripheral blood monocyte-derived macrophages (MDMs) can inhibit human immunodeficiency virus (HIV). We observed that the stimulation of MDMs with poly(dA:dT) or poly(dG:dC) (synthetic ligands for the DNA sensors) inhibited HIV infection and replication. MDMs treated with poly(dA:dT) or poly(dG:dC) expressed higher levels of both type I and type III IFNs than untreated cells. Activation of the DNA sensors in MDMs also induced the expression of the multiple intracellular anti-HIV factors, including IFN-stimulated genes (ISGs: ISG15, ISG56, Viperin, OAS2, GBP5, MxB, and Tetherin) and the HIV restriction microRNAs (miR-29c, miR-138, miR-146a, miR-155, miR-198, and miR-223). In addition, the DNA sensor activation of MDM upregulated the expression of the CC chemokines (RANTES, MIP-1α, MIP-1ß), the ligands for HIV entry coreceptor CCR5. These observations indicate that the cytosolic DNA sensors have a protective role in the macrophage intracellular immunity against HIV and that targeting the DNA sensors has therapeutic potential for immune activation-based anti-HIV treatment.

Exosomes Transport Anti-Human Immunodeficiency Virus Factors from Human Cervical Epithelial Cells to Macrophages.

The female reproductive tract (FRT) is a major site of HIV sexual transmission. As the outermost layer of cells in the FRT, the human cervical epithelial cells (HCEs) have direct contact with HIV or infected cells. Our early work showed that supernatant (SN) from TLR3-activated HCEs contain the antiviral factors that could potently inhibit HIV replication in macrophages. However, it remains to be determined how HCEs transport the anti-HIV factors to macrophages. This follow-up study examined the role of exosomes in HCE-mediated anti-HIV activity. We found that TLR3 activation of HCEs resulted in the release of exosomes that contained multiple IFN-stimulated genes (ISGs: ISG56, OAS1, MxA, and Mx2) and the HIV restriction microRNAs (miR-28, miR-29 family members, miR-125b, miR-150, miR-382, miR-223, miR-20a, and miR-198). The depletion of exosomes from SN of TLR3-activated HCEs diminished HCE-mediated anti-HIV activity in macrophages, indicating that HCE-derived exosomes are responsible for transporting the antiviral molecules to macrophages. These in vitro findings suggest a novel antiviral mechanism by which HCEs participate in the FRT innate immunity against HIV infection. Further in vivo studies are necessary in order to develop an exosome-based delivery system for prevention and treatment of HIV infection through sexual transmission.

CD4+ T cell depletion does not affect the level of viremia in chronically SHIVSF162P3N-infected Chinese cynomolgus monkeys.

Chronically SHIVSF162P3N-infected cynomolgus monkeys were used to determine the effects of the antibody-mediated acute CD4+ T cell depletion on viral load as well as on the immunological factors associated with disease progression. Compared with the control animals, CD4+ T cell-depleted animals with SHIV infection showed (i) little alteration in plasma viral load over the period of 22 weeks after the depletion; (ii) increased CD4+ T cell proliferation and turnover of macrophages at the early phase of the depletion, but subsequent decline to the basal levels; and (iii) little impact on the expression of the inflammatory cytokines and CC chemokines associated with disease progression. These findings indicate that the antibody-mediated acute CD4+ T cell depletion had minimal impact on plasma viral load and disease progression in chronically SHIVSF162P3N-infected cynomolgus monkeys. Future investigations are necessary to identify the key factor(s) related to the immune activation and macrophage infection during the CD4 deletion in chronic viral infection.

HIV infection suppresses TLR3 activation-mediated antiviral immunity in microglia and macrophages.

Monocytic-lineage cells in the central nervous system (CNS), including microglia and brain resident macrophages, are the key players in the CNS innate immunity against viral infections, including human immunodeficiency virus (HIV). However, these cells also serve as the major targets and reservoirs for HIV in the CNS. To address the question of how HIV can establish persistent infection in the target cells in the CNS, we examined whether HIV has the ability to counteract Toll-like receptor 3 (TLR3) activation-mediated antiviral immunity in microglia and macrophages. We observed that HIV latently infected microglial cells (HC69·5) expressed reduced levels of TLR3 and TLR3 activation-mediated interferons (IFN-α/ß and IFN-λ) as compared with the uninfected control cells (C20). In addition, HIV infection of primary human macrophages suppressed the expression of TLR3 and the IFNs. HIV infection also inhibited the expression of the antiviral IFN-stimulated genes (ISGs) and the HIV-restriction miRNAs. Mechanistically, HIV infection inhibited the phosphorylation of IFN regulatory factors (IRF3 and IRF7) and signal transducer and activator of transcription proteins (STAT1 and STAT3) in both HIV latently infected microglia and acutely infected macrophages. These findings provide previously unrecognized and sound mechanisms for HIV infection and persistence in the primary target and reservoir cells in the brain.

Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells.

AIMS: Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. METHODS: HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. RESULTS: Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. CONCLUSIONS: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

Heroin Abuse and/or HIV Infection Dysregulate Plasma Exosomal miRNAs.

Exosomes play an important role in cell-to-cell communication as they can transfer functional molecules such as microRNAs (miRNAs) from one cell to another, exerting biological and immunological functions. Here, we investigated the impact of HIV infection and/or heroin use on the expression of the miRNAs in plasma exosomes. We found that HIV infection or heroin use upregulated the majority (98%) of a panel of plasma exosomal miRNAs associated with immune regulation and inflammation. We also observed the enhanced effect of HIV infection and heroin use on some of these upregulated miRNAs. Our further investigation showed that the levels of four of neuro-inflammation-related miRNAs (146a, 126, 21, and let-7a) were higher in HIV-infected heroin users as compared with the control subjects. These findings indicate that the dysregulations of the plasma exosomal miRNAs support further studies to determine the role of the miRNAs in HIV and/or heroin use-mediated immune modulation and neuro-inflammation. Graphical abstract.

Poly(dA:dT) Suppresses HSV-2 Infection of Human Cervical Epithelial Cells Through RIG-I Activation.

Epithelial cells of the female reproductive tract (FRT) participate in the initial innate immunity against viral infections. Poly(dA:dT) is a synthetic analog of B form double-stranded (ds) DNA which can activate the interferon (IFN) signaling pathway-mediated antiviral immunity through DNA-dependent RNA Polymerase III. Here we investigated whether poly(dA:dT) could inhibit herpes simplex virus type 2 (HSV-2) infection of human cervical epithelial cells (End1/E6E7). We demonstrated that poly(dA:dT) treatment of End1/E6E7 cells could significantly inhibit HSV-2 infection. Mechanistically, poly(dA:dT) treatment of the cells induced the expression of the intracellular IFNs and the multiple antiviral IFN-stimulated genes (ISGs), including IFN-stimulated gene 15 (ISG15), IFN-stimulated gene 56 (ISG56), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 2 (OAS2), myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (Viperin), and guanylate binding protein 5 (GBP5). Further investigation showed that the activation of RIG-I was largely responsible for poly(dA:dT)-mediated HSV-2 inhibition and IFN/ISGs induction in the cervical epithelial cells, as RIG-I knockout abolished the poly(dA:dT) actions. These observations demonstrate the importance for design and development of AT-rich dsDNA-based intervention strategies to control HSV-2 mucosal transmission in FRT.

Morphine Withdrawal Enhances HIV Infection of Macrophages.

Opioid withdrawal recurs at high rates in opioid use disorder and compromises the immune system. In general, there are two types of opioid withdrawal: abrupt withdrawal (AW) and precipitated withdrawal (PW). In this study, we examined the effect of morphine AW or morphine PW on HIV infection of human blood monocyte-derived macrophages. We observed that both morphine AW and PW enhanced the susceptibility of macrophages to HIV infection. In addition, both AW and PW activated HIV replication in the latently infected myeloid cells (U1 and OM10.1). Investigation of mechanisms responsible for these observations showed that both AW and PW could inhibit the expression of multiple intracellular HIV inhibitory factors, including APOBE3G/F, SAMHD1, MX2, and HIV restriction microRNAs (miR-28, miR-125b, and miR-150) in macrophages. These findings provide additional evidence to support the notion that opioid use compromises the intracellular anti-HIV immunity and facilitates HIV infection and persistence in macrophages.

Isolation and characterization of spontaneously immortalized B-lymphocyte lines from HIV-infected patients with and without non-Hodgkin's Lymphoma.

Isolation of viable circulating tumor cells (CTC) holds the promise for improving screening, early diagnosis, and personalized treatment of lymphoma. In this study, we isolated and characterized spontaneously immortalized B-lymphocyte (SIBC) lines from HIV-infected patients with and without Non-Hodgkin's Lymphoma (AIDS-NHL). A total of 22 SIBC lines was isolated from peripheral blood mononuclear cells (PBMC) of HIV-infected patients with (n = 40) and without (n = 77) clinically detectable NHL, but not from healthy individuals (n = 34). Of these, 8 SIBC lines named HIV-SIBC were generated from HIV-infected patients without AIDS-NHL (10%, 8/77), while 14 SIBCs named AIDS-NHL-SIBC were from 13 of the AIDS-NHL patients (32.5%, 13/40). Among the 14 AIDS-NHL-SIBCs, 12 were derived from AIDS-NHL patients with poor prognoses (survival time less than 1 year). SIBCs displayed markers typical of memory B cells (CD3- CD20+ CD27+ ) with EBV infection. Moreover, AIDS-NHL-SIBCs were representative of CTC as evidenced by monoclonal Ig gene rearrangement, abnormal chromosomal karyotype, and the formation of xenograft tumors, while HIV-SIBCs generated harbored some features of tumor cells, none had the capacity of xenograft tumor formation, suggesting HIV-SIBC present the precursor of CTC. These results indicate that SIBCs is associated with poor prognosis in AIDS-NHL patients and can be isolated from HIV-infected patients with NHL and without NHL. This findings point to the need for further molecular characterization and functional studies of SIBCs, which may prove the value of SIBCs in the diagnosis, prognoses, and screening for NHL among HIV-infected patients.

IL-22 suppresses HSV-2 replication in human cervical epithelial cells.

Interleukin (IL)-22, a member of the IL-10 family, plays a role in antiviral immune responses to a number of viral infections. However, it is unclear whether IL-22 is involved in the mucosal immunity against herpes simplex virus 2 (HSV-2) infection in the female reproductive tract (FRT). In this study, we studied whether IL-22 could inhibit HSV-2 infection of human cervical epithelial cells (End1/E6E7 cells). We showed that End1/E6E7 cells express the functional IL-22 receptor complex (IL-22R1 and IL-10R2). When treated with IL-22, End1/E6E7 cells expressed the higher levels of IFN-stimulated genes (ISGs: ISG15, ISG56, OAS-1, OAS-2, and Mx2) than untreated cells. In addition, IL-22-treated cells produced higher levels of the tight junction proteins (ZO-1 and Occludin) than untreated cells. Mechanistically, IL-22 could activate the JAK/STAT signaling pathway by inducing the phosphorylation of STAT1 and STAT3. These observations indicate the potential of IL-22 as an anti-HSV-2 agent in the FRT mucosal innate immunity against HSV-2 infection.

Human Cervical Epithelial Cells Release Antiviral Factors and Inhibit HIV Replication in Macrophages.

The female reproductive tract is a major site of HIV sexual transmission. We here examined whether human cervical epithelial cells (HCEs) can be immunologically activated and produce antiviral factors against HIV. We demonstrated that HCEs (End1/E6E7 cells) possess the functional toll-like receptor (TLR)3 signaling system, which could be activated by Poly I:C and induce multiple cellular HIV restriction factors. The treatment of primary human macrophages with supernatant (SN) from TLR3-activated End1/E6E7 cell cultures resulted in HIV inhibition. This SN-mediated HIV inhibition was mainly through the induction of interferons (IFN)-ß and IFN-λs, as the antibodies to IFN-ß or IFN-λs receptor could effectively block the SN-mediated anti-HIV effect. Further studies showed that the incubation of macrophages with SN from the activated cervical epithelial cell cultures induced the expression of a number of IFN-stimulated genes (ISGs), including IFN-stimulated gene (ISG15), ISG56, 2', 5'-oligoadenylate synthetase 1 (OAS 1), OAS 2, Myxovirus Resistance A (MxA), MxB, and Guanylate-binding protein 5 (GBP5). In addition, TLR3-activated cells produced the CC chemokines [regulated on activation, normal T cell expressed and secreted (RANTES), Human macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1ß] the ligands of HIV entry co-receptor CCR5. These observations support further studies on HCEs as potentially crucial and alternative targets for immunological intervention to control and prevent HIV sexual transmission.

Multidrug-resistant tuberculosis (MDR-TB) strain infection in macaques results in high bacilli burdens in airways, driving broad innate/adaptive immune responses.

Tuberculosis (TB) has become the most deadly infectious diseases due to epidemics of HIV/AIDS and multidrug-resistant/extensively drug-resistant TB (MDR-/XDR-TB). Although person-to-person transmission contributes to MDR-TB, it remains unknown whether infection with MDR strains resembles infection with drug-sensitive (DS) TB strains, manipulating limited or broad immune responses. To address these questions, macaques were infected with MDR strain V791 and a drug-sensitive Erdman strain of TB. MDR bacilli burdens in the airway were significantly higher than those of the Erdman control after pulmonary exposure. This productive MDR strain infection upregulated the expression of caspase 3 in macrophages/monocytes and induced appreciable innate-like effector responses of CD3-negative lymphocytes and Ag-specific γδ T-cell subsets. Concurrently, MDR strain infection induced broad immune responses of T-cell subpopulations producing Th1, Th17, Th22, and CTL cytokines. Furthermore, MDR bacilli, like the Erdman strain, were capable of inducing typical TB disease characterized by weight loss, lymphocytopenia, and severe TB lesions. For the first time, our results suggest that MDR-TB infection acts like DS to induce high bacterial burdens in the airway (transmission advantage), innate/adaptive immune responses, and disease processes. Because nonhuman primates are biologically closer to humans than other species, our data may provide useful information for predicting the effects of primary MDR strain infection after person-to-person transmission. The findings also support the hypothesis that a vaccine or host-directed adjunctive modality that is effective for drug-sensitive TB is likely to also impact MDR-TB.

Bowman‒Birk Inhibitor Suppresses Herpes Simplex Virus Type 2 Infection of Human Cervical Epithelial Cells.

The Bowman‒Birk inhibitor (BBI), a protease inhibitor derived from soybeans, has been extensively studied in anti-tumor and anti-inflammation research. We recently reported that BBI has an anti-HIV-1 property in primary human macrophages. Because HSV-2 infection plays a role in facilitating HIV-1 sexual transmission, we thus examined whether BBI has the ability to inhibit HSV-2 infection. We demonstrated that BBI could potently inhibit HSV-2 replication in human cervical epithelial cells (End1/E6E7). This BBI-mediated HSV-2 inhibition was partially through blocking HSV-2-mediated activation of NF-κB and p38 MAPK pathways. In addition, BBI could activate the JAK/STAT pathway and enhance the expression of several antiviral interferon-stimulated genes (ISGs). Furthermore, BBI treatment of End1/E6E7 cells upregulated the expression of tight junction proteins and reduced HSV-2-mediated cellular ubiquitinated proteins' degradation through suppressing the ubiquitin‒proteasome system. These observations indicate that BBI may have therapeutic potential for the prevention and treatment of HSV-2 infections.

Alcohol-induced autophagy via upregulation of PIASy promotes HCV replication in human hepatoma cells.

Both alcohol and hepatitis C virus (HCV) infection could induce cellular autophagy in liver cells, which is considered to be essential for productive HCV replication. However, whether alcohol-induced autophagy is involved in the pathogenesis of HCV infection is still poorly understood. Alcohol treatment could induce autophagy in Huh7 cells (a hepatoma cell line that supports HCV JFH-1 replication), evidenced by the increase of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the decrease of p62 level in alcohol-treated cells compared with control cells. Alcohol treatment also significantly increased PIASy (a member of the PIAS family) expression, which can act as a SUMO (small ubiquitin-like modifier protein) E3 ligase to regulate a broader range of cellular processes including autophagy. Overexpression or the silencing expression of PIASy in alcohol-treated Huh7 cells could increase or decrease autophagic activation caused by alcohol treatment, respectively, and thus affect HCV replication correspondingly. In the absence of alcohol, overexpression or silencing expression of PIASy increase or decrease the level of cellular autophagy, judged by the changes of LC3B-II and p62 levels in the presence or absence of chloroquine (CQ), a lysosome inhibitor. More importantly, in the presence of 3-methyladenine (3-MA), an inhibitor in the early stage of autophagy, the effects of overexpression or silencing expression of PIASy on HCV replication were largely blocked. Furthermore, PIASy could selectively drive the accumulation of SUMO1-conjugated proteins, along with upregulation of the expression of several important autophagy factors, including ATG7 and ATG5-ATG12. In conclusion, alcohol promotes HCV replication through activation of autophagy in Huh7 cells, which partly attributes to its induction of PIASy expression. PIASy-enhanced accumulation of SUMO1-conjugated proteins may contribute to its inducing effect of autophagy. Our findings provide a novel mechanism for the action of alcohol-promoting HCV replication in the context of cellular autophagy.

IFN-λ4 inhibits HIV infection of macrophages through signalling of IFN-λR1/IL-10R2 receptor complex.

The recently discovered IFN-λ4 has been found to have antiviral activity against several viruses. However, it's unknown whether IFN-λ4 can inhibit HIV infection. Here, we show that IFN-λ4 could suppress HIV infection of macrophages. This IFN-λ4-mediated HIV inhibition was compromised by the antibodies against IFN-λ receptor complex, IFN-λR1/IL-10R2. IFN-λ4 enhanced the phosphorylation of STAT1, and induced antiviral interferon-stimulated genes. These findings indicated that IFN-λ4 can inhibit HIV via JAK/STAT signalling pathway.

Human Intestinal Epithelial Cells Release Antiviral Factors That Inhibit HIV Infection of Macrophages.

As a rich source of CD4+ T cells and macrophages, the gastrointestinal (GI) tract is a major target site for HIV infection. The interplay between GI-resident macrophages and intestinal epithelial cells (IECs) constitutes an important element of GI innate immunity against pathogens. In this study, we investigated whether human IECs have the ability to produce antiviral factors that can inhibit HIV infection of macrophages. We demonstrated that IECs possess functional toll-like receptor 3 (TLR3), the activation of which resulted in induction of key interferon (IFN) regulatory factors (IRF3 and IRF7), IFN-ß, IFN-λ, and CC chemokines (MIP-1α, MIP-1ß, RANTES), the ligands of HIV entry co-receptor CCR5. In addition, TLR3-activated IECs release exosomes that contained the anti-HIV factors, including IFN-stimulated genes (ISGs: ISG15, ISG56, MxB, OAS-1, GBP5, and Viperin) and HIV restriction miRNAs (miRNA-17, miRNA-20, miRNA-28, miRNA-29 family members, and miRNA-125b). Importantly, treatment of macrophages with supernatant (SN) from the activated IEC cultures inhibited HIV replication. Further studies showed that IEC SN could also induce the expression of antiviral ISGs and cellular HIV restriction factors (Tetherin and APOBEC3G/3F) in HIV-infected macrophages. These findings indicated that IECs might act as an important element in GI innate immunity against HIV infection/replication.

Alcohol attenuates anti-HCV function of IFN-λ1 through up-regulation of PLASy expression in human hepatic cells.

Alcohol could compromise the anti-hepatitis C virus (HCV) function of interferon-alpha (IFN-α). However, little information is available about the effect of alcohol on interferon-lambda (IFN-λ, type III IFN), a novel candidate for development of therapy for HCV infection. Huh7 cells were infected with HCV JFH-1 virus, then treated with alcohol, and/or IFN-λ1. RT-PCR and Western blot were used to detect the levels of HCV and key cellular factors. Overexpression or silencing expression was performed to verify the role of key factors in alcohol-attenuated anti-HCV function of IFN-λ1. Alcohol treatment compromised anti-HCV effect of IFN-λ1 in HCV JFH-1-infected Huh7 cells, evidenced by the significantly increased levels of HCV RNA, and HCV core protein in alcohol-/IFN-λ1-treated cells compared to cells with IFN-λ1 treatment alone. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol enhanced the expression of protein inhibitor of activated STAT (PIASy). Overexpression of PIASy compromised anti-HCV ability of IFN-λ1, whereas silencing expression of PIASy partly restored the alcohol-attenuated anti-HCV effect of IFN-λ1. More importantly, overexpression of PIASy significantly down-regulated the level of IFN-λ1-indcued phosphorylation of STAT1 (p-STAT1), an important adaptor in IFN-λ pathway, as well as reduced the expression of IFN-λ1-induced IFN-stimulated genes 56 (ISG56), and myxovirus resistance 1 (Mx1), two antiviral effectors in in IFN-λ pathway. These findings indicate that alcohol, through inducing the expression of negative regulator in IFN-λ pathway, inhibits IFN-λ-mediated anti-HCV action in human hepatic cells, which may lead to the poor efficacy of IFN-λ-based therapy against HCV infection.

GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors.

Although it has been shown that some mannose-binding lectins (MBLs) exhibit significant activity against HIV infection, little is known about whether N-acetylgalactosamine (GalNAc)-binding lectins have the ability to inhibit HIV infection. Here, we demonstrate that a soybean-derived lectin (SBL) with GalNAc-binding affinity could potently suppress HIV infection of macrophages in a dose-dependent fashion. Unlike the MBLs, which block HIV only through binding to the glycosylated envelope proteins (gp120 and gp41) of the virus, SBL inhibited HIV at multiple steps of the virus infection/replication cycle. SBL could activate the beta interferon (IFN-ß)-STAT signaling pathway, resulting in the upregulation of a number of antiviral interferon-stimulated genes (ISGs) in macrophages. In addition, SBL treatment of macrophages induced the production of C-C chemokines, which bind to HIV entry coreceptor CCR5. Deglycosylation of cell surface galactosyl moieties or presaturation of GalNAc-binding capacity could compromise SBL-mediated induction of the antiviral factors. Furthermore, SBL exerted its anti-HIV activity in the low nanomolar range with no mitogenic effect on CD4+ T cells, a major advantage in the development of SBL as a potential anti-HIV agent compared with MBLs. These data indicate a necessity to further investigate SBL as an alternative and cost-effective anti-HIV natural product.IMPORTANCE Mannose-binding lectins (MBLs) can block the attachment of HIV to target cells and have been suggested as anti-HIV microbicides. However, the mitogenic effect of MBLs on CD4+ T cells limits this potential in clinical settings. Lectins with galactose (Gal)- or N-acetylgalactosamine (GalNAc)-binding specificity are another important category of carbohydrate-binding proteins (CBP). Compared to high-mannose N-linked glycans, GalNAc-type glycans present much less in HIV gp120 or gp41 glycosylation. Here, we demonstrate that GalNAc-specific soybean lectin (SBL) triggers antiviral signaling via recognition of the cell surface galactosyl group of macrophages, which results in the suppression of HIV at multiple steps. More importantly, SBL has no mitogenic effect on the activation of CD4+ T cells, a major advantage in the development of Gal/GalNAc-specific lectins as naturopathic anti-HIV agents.