Transcriptional regulation of the HIV-1 inhibitory factor human mannose receptor 1 by the myeloid-specific transcription factor PU.1.

HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.

Human Mannose Receptor 1 Attenuates HIV-1 Infectivity in a Virus Isolate-Specific Manner.

Human mannose receptor 1 (hMRC1) is a transmembrane glycoprotein that belongs to the C-type lectin family and is expressed on the surface of most tissue macrophages. hMRC1 contributes to the binding and transmission of HIV-1 and is involved in the endocytic uptake of HIV-1 for subsequent antigen presentation. We previously reported that hMRC1 functions as an antiviral factor by inhibiting virus release through a BST-2-like mechanism. The inhibition of virus release was not virus isolate-specific and, surprisingly, was not Env-dependent. We now report on another hMRC1 antiviral function that affects the infectivity of viral particles. Unlike its effect on virus release, the inhibition of viral infectivity by hMRC1 was virus isolate-specific. An analysis of chimeric Env revealed that the Env V3 region was a critical determinant for the inhibitory effect of hMRC1. Of note, exogenously expressed hMRC1 was packaged into viral particles in an Env-independent manner. Co-immunoprecipitation studies revealed a strong interaction of the hMRC1-sensitive NL43 Env with hMRC1, while the hMRC1-insensitive Envs of AD8 and 49.5 isolates interacted poorly if at all with hMRC1. An analysis of a panel of Transmitted/Founder (T/F) viruses revealed that all of them were R5-tropic, and more than half of them were inhibited by hMRC1. The detailed mechanism of how hMRC1 inhibits viral infectivity remains to be investigated. However, the high-affinity binding of hMRC1 to Env may cause a conformational change around the Env V3 region or obstruct the Env V3 region and may make it inaccessible for subsequent interaction with the coreceptor during virus entry.

HIV-1 Vpr Induces Degradation of Gelsolin, a Myeloid Cell-Specific Host Factor That Reduces Viral Infectivity by Inhibiting the Expression and Packaging of the HIV-1 Env Glycoprotein.

Gelsolin (GSN) is a structural actin-binding protein that is known to affect actin dynamics in the cell. Using mass spectrometry, we identified GSN as a novel Vpr-interacting protein. Endogenous GSN protein was expressed at detectable levels in monocyte-derived macrophages (MDM) and in THP-1 cells, but it was undetectable at the protein level in other cell lines tested. The HIV-1 infection of MDM was associated with a reduction in GSN steady-state levels, presumably due to the Vpr-induced degradation of GSN. Indeed, the coexpression of GSN and Viral protein R (Vpr) in transiently transfected HEK293T cells resulted in the Vpr-dependent proteasomal degradation of GSN. This effect was observed for Vprs from multiple virus isolates. The overexpression of GSN in HEK293T cells had no effect on Gag expression or particle release, but it reduced the expression and packaging of the HIV-1 envelope (Env) glycoprotein and reduced viral infectivity. An analysis of the HIV-1 splicing patterns did not reveal any GSN-dependent differences, suggesting that the effect of GSN on Env expression was regulated at a posttranscriptional level. Indeed, the treatment of transfected cells with lysosomal inhibitors reversed the effect of GSN on Env stability, suggesting that GSN reduced Env expression via enhanced lysosomal degradation. Our data identify GSN as a macrophage-specific host antiviral factor that reduces the expression of HIV-1 Env. IMPORTANCE Despite dramatic progress in drug therapies, HIV-1 infection remains an incurable disease that affects millions of people worldwide. The virus establishes long-lasting reservoirs that are resistant to currently available drug treatments and allow the virus to rebound whenever drug therapy is interrupted. Macrophages are long-lived cells that are relatively insensitive to HIV-1-induced cytopathicity and thus could contribute to the viral reservoir. Here, we identified a novel host factor, gelsolin, that is expressed at high levels in macrophages and inhibits viral infectivity by modulating the expression of the HIV-1 Env glycoprotein, which is critical in the spread of an HIV-1 infection. Importantly, the viral protein Vpr induces the degradation of gelsolin and thus counteracts its antiviral activity. Our study provides significant and novel insights into HIV-1 virus-host interactions and furthers our understanding of the importance of Vpr in HIV-1 infection and pathogenesis.

4-phenylquinoline-8-amine induces HIV-1 reactivation and apoptosis in latently HIV-1 infected cells.

Combinational antiretroviral therapy (cART) dramatically suppresses the viral load to undetectable levels in human immunodeficiency virus (HIV)-infected patients. However, HIV-1 reservoirs in CD4+T cells and myeloid cells, which can evade cART and host antiviral immune systems, are still significant obstacles to HIV-1 eradication. The "Shock and Kill" approach using latently-reversing agents (LRAs) is therefore currently developing strategies for effective HIV-1 reactivation from latency and inducing cell death. Here, we performed small-molecular chemical library screening with monocytic HIV-1 latently-infected model cells, THP-1 Nluc #225, and identified 4-phenylquinoline-8-amine (PQA) as a novel LRA candidate. PQA induced efficient HIV-1 reactivation in combination with PKC agonists including Prostratin and showed a similar tendency for HIV-1 activation in primary HIV-1 reservoirs. Furthermore, PQA induced killing of HIV-1 latently-infected cells. RNA-sequencing analysis revealed PQA had different functional mechanisms from PKC agonists, and oxidative stress-inducible genes including DDIT3 or CTSD were only involved in PQA-mediated cell death. In summary, PQA is a potential LRA lead compound that exerts novel functions related to HIV-1 activation and apoptosis-mediated cell death to eliminate HIV-1 reservoirs.

The Myeloid-Specific Transcription Factor PU.1 Upregulates Mannose Receptor Expression but Represses Basal Activity of the HIV-LTR Promoter.

Human mannose receptor 1 (MRC1) is a cell surface receptor expressed in macrophages and other myeloid cells that inhibits human immunodeficiency virus type 1 (HIV-1) particle release by tethering virions to producer cell membranes. HIV-1 counteracts MRC1 expression by inhibiting mrc1 transcription. Here, we investigated the mechanism of MRC1 downregulation in HIV-1-infected macrophages. We identified the myeloid cell-specific transcription factor PU.1 as critical for regulating MRC1 expression. In the course of our study, we recognized a complex interplay between HIV-1 Tat and PU.1 transcription factors: Tat upregulated HIV-1 gene expression but inhibited mrc1 transcription, whereas PU.1 inhibited HIV-1 transcription but activated MRC1 expression. Disturbing this equilibrium by silencing PU.1 resulted in increased HIV-1 gene expression and reduced MRC1 promoter activity. Our study identified PU.1 as a central player in transcriptional control, regulating a complex interplay between viral and host gene expression in HIV-infected macrophages. IMPORTANCE HIV-1 replication in primary human cells depends on the activity of virus-encoded proteins but also involves cellular factors that can either promote (viral dependency factors) or inhibit (host restriction factors) virus replication. In previous work, we identified human MRC1 as a macrophage-specific host restriction factor that inhibits the detachment of viral particles from infected cells. Here, we report that HIV-1 counteracts this effect of MRC1 by imposing a transcriptional block on cellular MRC1 gene expression. The transcriptional inhibition of the MRC1 gene is accomplished by Tat, an HIV-1 factor whose best-described function actually is the enhancement of HIV-1 gene expression. Thus, HIV-1 has evolved to use the same protein for (i) activation of its own gene expression while (ii) inhibiting expression of MRC1 and other host factors.

Evaluation of the Geenius HIV 1/2 confirmatory assay for HIV-2 samples isolated in Japan.

BACKGROUND: Although the number of HIV-2-infected individuals is quite low in Japan, at least three groups of HIV-2 (A, B and CRF01_AB) have been detected thus far. In particular, CRF01_AB HIV-2 cases have been found only in limited areas, Cote d'Ivoire and Japan. Here, we demonstrate that Geenius HIV 1/2 Confirmatory Assay (Geenius, Bio-Rad Laboratories) is able to detect HIV-2 samples, including groups A, B and CRF01_AB, isolated in Japan. STUDY DESIGN: A total of 57 plasma samples, including three panels (Ⅰ: HIV-2-positive samples [n=9], Ⅱ: HIV-1 infection with HIV-2 antibody cross-reactivity samples [n=37], and Ⅲ: HIV negative with biological false-positive HIV-2 samples [n=11]) were tested by Geenius. RESULTS: Geenius determined Panel I to be "HIV-2 positive with/without HIV-1 cross-reactivity (n=4, respectively)", including HIV-2 group A and CRF01_AB. In the case with HIV-2 group B, all bands were detected, resulting in a Geenius interpretation of "HIV positive untypable". Geenius classified Panels II and III as "HIV-1 positive (n=37)" or "HIV negative (n=9)", "HIV indeterminate (n=1)" and "HIV-2 indeterminate (n=1)", suggesting 95.8% HIV-2 differentiation by Geenius. CONCLUSIONS: With Geenius, there were fewer false-positives for HIV-1/-2 negativity and fewer cross-reactions with HIV-2 among HIV-1-positive samples. Additionally, the assay could detect HIV-2 genetic group CRF01_AB. Geenius can be expected to be a useful diagnostic tool that is an alternative to conventional Western blotting.

[Genomics and epidemiology of SARS-CoV-2 lineage].

Coronavirus disease 2019（COVID-19）is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）．It had been first identified in Wuhan at the end of 2019 and the spread of SARS-CoV-2 variants has become a crucial issue worldwide. WHO categorized SARS-CoV-2 variants as "Variants of Concern; VOCs" and "Variants of Interest; VOIs" based on transmissibility, disease severity or their susceptibility to vaccines. Especially, the spread of SARS-CoV-2 variant categorized in VOCs, such as B.1.1.7 and B.1.617.2, has been a serious concern worldwide. In Japan, in addition to the B.1.1.214/B.1.1.284 variants, the B.1.1.7 variant has been rapidly spreading in Osaka and Tokyo. The B.1.617 variant was first identified in April 2021 in a patient who was under domestic quarantine and cases of community-acquired infections of the B.1.617.2 variant were first observed in May 2021. Amino acid changes in the spike protein, such as the N501Y, E484K or L452R mutations mainly observed in VOCs in addition to the D614G mutation are thought to affect the transmissibility and immune escape of the virus as well as the disease severity and this may be contributory to the rapid spread of SARS-CoV-2 variants. Now, several SARS-CoV-2 variants with additional mutations are continuously emerging worldwide and the prevailing SARS-CoV-2 variants are rapidly changing.

Inhibition of Vif-Mediated Degradation of APOBEC3G through Competitive Binding of Core-Binding Factor Beta.

Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFß) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFß. Only mutants that retained the ability to bind CBFß exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFß and wild-type Vif. Furthermore, overexpression of CBFß counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFß as the key player involved in D/N interference by Vif.IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFß), which is expressed in cells in limiting quantities. Overexpression of CBFß neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFß, phenocopied the D/N Vif phenotype by sequestering endogenous CBFß. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.

Discovery of 4-oxoquinolines, a new chemical class of anti-HIV-1 compounds.

Antiretroviral therapy (ART) against HIV-1 infection offers the promise of controlling disease progression and prolonging the survival of HIV-1-infected patients. However, even the most potent ART regimens available today cannot cure HIV-1. Because patients will be exposed to ART for many years, physicians and researchers must anticipate the emergence of drug-resistant HIV-1, potential adverse effects of the current drugs, and need for future drug development. In this study, we screened a small-molecule compound library using cell-based anti-HIV-1 assays and discovered a series of novel anti-HIV-1 compounds, 4-oxoquinolines. These compounds exhibited potent anti-HIV-1 activity (EC50 < 0.1 µM) with high selectivity indexes (CC50/EC50 > 2500) and favorable pharmacokinetic profiles in mice. Surprisingly, our novel compounds have a chemical backbone similar to the clinically used integrase (IN) strand transfer inhibitor (INSTI) elvitegravir, although they lack the crucial 3-carboxylate moiety needed for the common INSTI diketo motif. Indeed, the new 4-oxoquinoline derivatives have no detectable INSTI activity. In addition, various drug-resistant HIV-1 strains did not display cross resistance to these compounds. Interestingly, time-of-addition experiments indicated that the 4-oxoquinoline derivative remains its anti-HIV-1 activity even after the viral integration stage. Furthermore, the compounds significantly suppressed p24 antigen production in HIV-1 latently infected cells exposed with tumor necrosis factor alpha. These findings suggest that our 4-oxoquinoline derivatives with no 3-carboxylate moiety may become novel lead compounds in the development of anti-HIV-1 drugs.

Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

Robust Enhancement of Lentivirus Production by Promoter Activation.

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

HIV-1 Vpr and p21 restrict LINE-1 mobility.

Long interspersed element-1 (LINE-1, L1) composes ∼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.

Mannose Receptor 1 Restricts HIV Particle Release from Infected Macrophages.

Human mannose receptor 1 (hMRC1) is expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages (MDMs) and is involved in the endocytic uptake of HIV-1 into these cells. Here, we identify hMRC1 as an antiviral factor that inhibits virus release through a bone marrow stromal antigen 2 (BST-2)-like mechanism. Virions produced in the presence of hMRC1 accumulated in clusters at the cell surface but were fully infectious. HIV-1 counteracted the effect by transcriptional silencing of hMRC1. The effect of hMRC1 was not virus isolate specific. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1 antiviral activity, suggesting the involvement of additional cellular factor(s) in the process. Our data reveal an antiviral mechanism that is active in primary human macrophages and is counteracted by HIV-1 through downregulation of hMRC1.

Suppressor of cytokine signaling 1 counteracts rhesus macaque TRIM5α-induced inhibition of human immunodeficiency virus type-1 production.

Old world monkey TRIM5α is a host factor that restricts human immunodeficiency virus type-1 (HIV-1) infection. Previously, we reported that rhesus macaque TRIM5α (RhTRIM5α) restricts HIV-1 production by inducing degradation of precursor Gag. Since suppressor of cytokine signaling 1 (SOCS1) is known to enhance HIV-1 production by rescuing Gag from lysosomal degradation, we examined if SOCS1 is involved in RhTRIM5α-mediated late restriction. Over-expression of SOCS1 restored HIV-1 production in the presence of RhTRIM5α to a level comparable to that in the absence of RhTRIM5α in terms of titer and viral protein expression. Co-immunoprecipitation studies revealed that SOCS1 physically interacted with RhTRIM5α. Over-expression of SOCS1 affected RhTRIM5α expression in a dose-dependent manner, which was not reversed by proteasome inhibitors. In addition, SOCS1 and RhTRIM5α were detected in virus-like particles. These results suggest that SOCS1 alleviates RhTRIM5α-mediated regulation in the late phase of HIV-1 life cycle probably due to the destabilization of RhTRIM5α.