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

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

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

Enterokinase and IAV Infection

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

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

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

Lamellarin α20-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 α20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV

CXCR4-Tropic, But Not CCR5-Tropic, Human Immunodeficiency Virus Infection Is Inhibited by the Lipid Raft-Associated Factors, Acyclic Retinoid Analogs, and Cholera Toxin B Subunit

Development of an effective low-cost anti-acquired immunodeficiency syndrome (AIDS) drugs is needed for treatment of AIDS patients in developing countries. Host cell lipid raft microdomains, which are enriched with cholesterol, glycolipids, ceramide, and gangliosides, are important for human immunodeficiency virus type 1 (HIV-1) entry. Retinoid analogs have been shown to modulate ceramide levels in the cell membrane, while cholera toxin B subunit (CT-B) specifically binds to the ganglioside GM1. In this study, we found that the acyclic retinoid analogs geranylgeranoic acid (GGA) and NIK-333 as well as CT-B efficiently attenuate CXCR4-tropic, but not CCR5-tropic, HIV-1 vector infection. We also found that GGA and NIK-333 suppress CXCR4-tropic HIV-1 infection by attenuating CXCR4 expression. CT-B also attenuated CXCR4-tropic HIV-1 infection, but did not suppress CXCR4 expression. These results suggest a distinct role for lipid raft microdomains in CXCR4- and CCR5-tropic HIV-1 infections and illuminate novel agents for the development of AIDS therapy

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

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse ( Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1