Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport.

Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.

Highly Neutralizing COVID-19 Convalescent Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters.

Despite various attempts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients with COVID-19 convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly neutralizing COVID-19 convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing activity-determined convalescent plasmas, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in a significant reduction of viral titers in lungs by up to 32-fold compared to the viral titers in hamsters receiving control nonneutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs more than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using microcomputerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19 convalescent plasmas variably contained antibodies against SARS-CoV-2 components, including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent neutralizing activity-confirmed COVID-19 convalescent plasmas would be efficacious in treating patients with COVID-19. IMPORTANCE Convalescent plasmas obtained from patients who recovered from a specific infection have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized controlled clinical trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94 to 95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly neutralizing effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally induced lung lesions. The present data provide a proof of concept that the presence of highly neutralizing antibody in COVID-19 convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.

An outbreak of a novel lineage of dengue virus 2 in Vietnam in 2022.

In 2022, a large dengue outbreak was reported in Vietnam, where dengue was endemic. A total of 1889 acute-phase serum samples were collected from patients with suspected dengue at Vung Tau General Hospital, the core hospital in Vung Tau Province, southern Vietnam. Among the 1889 samples analyzed for laboratory confirmation of dengue virus (DENV) infection, 339 positive cases were identified, of which 130 were primary infections and 209 were secondary infections. DENV-2 was the dominant serotype in both primary and secondary infection groups. Phylogenetic analysis based on sequences of the envelope protein-coding region revealed the emergence of a new DENV-2 lineage during this outbreak.

Preserved SARS-CoV-2 neutralizing IgG activity of in-house manufactured COVID-19 convalescent plasma.

PURPOSE: In the current study, we aimed to evaluate the neutralizing IgG activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the coagulation factors of convalescent plasmas which we manufactured in-house without a fast-freezing technique. METHODS: We collected plasmas from eligible participants who had confirmed certain titers of neutralizing antibodies. The plasmas were frozen and stored in the ordinary biofreezer without a fast-freezing function. The purified-IgG neutralizing activity of 20 samples from 19 participants and the coagulation factors of 49 samples from 40 participants were evaluated before and after freezing. RESULTS: Purified-IgG maintained its neutralizing activities, with the median 50 % inhibitory concentration (IC50) of 10.11 mg/ml (IQR 6.53-18.19) before freezing and 8.90 m g/ml (IQR 6.92-28.27) after thawing (p = 0.956). On the contrary, fibrinogen and factor Ⅷ decreased significantly after freezing and thawing in our environment. No significant temperature deviation was observed during the storage period. CONCLUSION: Neutralizing IgG activity, which largely contributes to the antiviral activity of convalescent plasma, did not change through our in-house manufacturing, without fastfreezing and storage conditions for more than 200 days. Ordinary freezers without the fast-freezing function are suitable enough to manufacture and store convalescent plasmas. Hospitals or facilities without specified resources could easily collect and store convalescent plasmas in case of upcoming emerging or re-emerging infectious diseases on-demand with appropriate neutralizing antibody levels measurements.

Efficacy of convalescent plasma therapy for COVID-19 in Japan: An open-label, randomized, controlled trial.

BACKGROUND: Convalescent plasma is a potential therapeutic option for patients with coronavirus disease 2019 (COVID-19). Despite its use for treating several viral infections, we lack comprehensive data on its efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We conducted a multicenter, open-label, randomized controlled trial of convalescent plasma therapy with high neutralizing activity against SARS-CoV-2 in high-risk patients within five days after the onset of COVID-19 symptoms. The primary endpoint was the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs from days 0-5. RESULTS: Between February 24, 2021, and November 30, 2021, 25 patients were randomly assigned to either convalescent plasma (n = 14) or standard of care (n = 11) groups. Four patients discontinued their allocated convalescent plasma, and 21 were included in the modified intention-to-treat analysis. The median interval between the symptom onset and plasma administration was 4.5 days (interquartile range, 3-5 days). The primary outcome of the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs did not significantly differ between days 0-5 (1.2 log10 copies/mL in the convalescent plasma vs. 1.2 log10 copies/mL in the standard of care (effect estimate, 0.0 [95% confidence interval, -0.8-0.7]; P = 0.94)). No deaths were observed in either group. CONCLUSIONS: The early administration of convalescent plasma with high neutralizing activity did not contribute to a decrease in the viral load within five days compared with the standard of care alone.

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient.

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection.

Has COVID-19 suppressed dengue transmission in Nepal?

Following the report of the first COVID-19 case in Nepal on 23 January 2020, three major waves were documented between 2020 and 2021. By the end of July 2022, 986 596 cases of confirmed COVID-19 and 11 967 deaths had been reported and 70.5% of the population had received at least two doses of a COVID-19 vaccine. Prior to the pandemic, a large dengue virus (DENV) epidemic affected 68 out of 77 districts, with 17 932 cases and six deaths recorded in 2019. In contrast, the country's Epidemiology and Disease Control Division reported 530 and 540 dengue cases in the pandemic period (2020 and 2021), respectively. Furthermore, Kathmandu reported just 63 dengue cases during 2020 and 2021, significantly lower than the 1463 cases reported in 2019. Serological assay showed 3.2% positivity rates for anti-dengue immunoglobulin M antibodies during the pandemic period, contrasting with 26.9-40% prior to it. Real-time polymerase chain reaction for DENV showed a 0.5% positive rate during the COVID-19 pandemic which is far lower than the 57.0% recorded in 2019. Continuing analyses of dengue incidence and further strengthening of surveillance and collaboration at the regional and international levels are required to fully understand whether the reduction in dengue incidence/transmission were caused by movement restrictions during the COVID-19 pandemic.

Antibody titers and neutralizing activity in cases of COVID-19 after a single dose of vaccination.

Vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy in preventing the onset of disease. However, the immune response to infection immediately after the first vaccination remains unknown. We examined the anti-SARS-CoV-2-binding-antibody titers and neutralizing activity in patients who developed coronavirus disease 2019 after the first vaccination. The amount of anti-SARS-CoV-2-binding antibodies and neutralizing activity drastically increased from the first to the second collection. Our results may provide important data on the course of immune response following vaccination.

Successful use of casirivimab/imdevimab anti-spike monoclonal antibodies to enhance neutralizing antibodies in a woman on anti-CD20 treatment with refractory COVID-19.

Management of COVID-19 patients with humoral immunodeficiency is challenging. We describe a woman with COVID-19 with multiple relapses due to anti-CD20 monoclonal antibody treatment. She was successfully treated with casirivimab/imdevimab and confirmed to have neutralizing antibodies. This case suggests that monoclonal antibodies have therapeutic and prophylactic value in patients with humoral immunodeficiency.

[A Case of Suspected Lynch Syndrome Due to Radical Resection for Descending Colon Cancer and Renal Pelvic Cancer].

A 60-year-old woman was admitted on account of presenting with bloody stools. She had a history of endometrial cancer surgery. Family history revealed 3 colorectal cancer cases among the first or second relatives. Colonoscopy and contrast- enhanced computed tomography revealed descending colon cancer and left renal pelvic cancer. We performed partial resection of the descending/transverse colon with D3 lymph node dissection and total resection of the left kidney and ureter with curative intent. Postoperative pathological diagnosis revealed descending colon cancer(pT4bN0M1c, pStage Ⅳc)and left renal pelvic cancer (T1N0M0, Stage Ⅰ). In this case, Lynch syndrome was suspected based on the family history and medical history. The clinical findings were consistent with Amsterdam Criteria Ⅱ. The microsatellite instability(MSI)test result was MSI-H and the BRAF genetic test result showed a wild type. Immunohistochemical staining of descending colon cancer tissue showed loss of expression of MSH2 and MSH6 proteins. Genetic counseling was provided because Lynch syndrome was strongly suspected. Capecitabine plus oxaliplatin therapy was performed for 6 months for descending colon cancer. Nine months postoperatively, the patient remained recurrence-free for both colon cancer and renal pelvic cancer. We report a case of suspected Lynch syndrome triggered by double cancer of the descending colon and renal pelvis.

The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis.

While it is well appreciated that late domains in the viral matrix proteins are crucial to mediate efficient virus budding, little is known about roles of late domains in the viral nucleocapsid proteins. Here, we characterized the functional relevance of a YxxL motif with potential late-domain function in the Ebola virus nucleocapsid protein VP24. Mutations in the YxxL motif had two opposing effects on the functions of VP24. On the one hand, the mutation affected the regulatory function of VP24 in viral RNA transcription and replication, which correlated with an increased incorporation of minigenomes into released transcription- and replication-competent virus-like particles (trVLPs). Consequently, cells infected with those trVLPs showed higher levels of viral transcription. On the other hand, mutations of the YxxL motif greatly impaired the intracellular transport of nucleocapsid-like structures (NCLSs) composed of the viral proteins NP, VP35, and VP24 and the length of released trVLPs. Attempts to rescue recombinant Ebola virus expressing YxxL-deficient VP24 failed, underlining the importance of this motif for the viral life cycle.IMPORTANCE Ebola virus (EBOV) causes a severe fever with high case fatality rates and, so far, no available specific therapy. Understanding the interplay between viral and host proteins is important to identify new therapeutic approaches. VP24 is one of the essential nucleocapsid components and is necessary to regulate viral RNA synthesis and condense viral nucleocapsids before their transport to the plasma membrane. Our functional analyses of the YxxL motif in VP24 suggested that it serves as an interface between nucleocapsid-like structures (NCLSs) and cellular proteins, promoting intracellular transport of NCLSs in an Alix-independent manner. Moreover, the YxxL motif is necessary for the inhibitory function of VP24 in viral RNA synthesis. A failure to rescue EBOV encoding VP24 with a mutated YxxL motif indicated that the integrity of the YxxL motif is essential for EBOV growth. Thus, this motif might represent a potential target for antiviral interference.

Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport.

The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions. Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.

Novel Protease Inhibitors Containing C-5-Modified bis-Tetrahydrofuranylurethane and Aminobenzothiazole as P2 and P2' Ligands That Exert Potent Antiviral Activity against Highly Multidrug-Resistant HIV-1 with a High Genetic Barrier against the Emergence of Drug Resistance.

Combination antiretroviral therapy has achieved dramatic reductions in the mortality and morbidity in people with HIV-1 infection. Darunavir (DRV) represents a most efficacious and well-tolerated protease inhibitor (PI) with a high genetic barrier to the emergence of drug-resistant HIV-1. However, highly DRV-resistant variants have been reported in patients receiving long-term DRV-containing regimens. Here, we report three novel HIV-1 PIs (GRL-057-14, GRL-058-14, and GRL-059-14), all of which contain a P2-amino-substituted-bis-tetrahydrofuranylurethane (bis-THF) and a P2'-cyclopropyl-amino-benzothiazole (Cp-Abt). These PIs not only potently inhibit the replication of wild-type HIV-1 (50% effective concentration [EC50], 0.22 nM to 10.4 nM) but also inhibit multi-PI-resistant HIV-1 variants, including highly DRV-resistant HIVDRVRP51 (EC50, 1.6 nM to 30.7 nM). The emergence of HIV-1 variants resistant to the three compounds was much delayed in selection experiments compared to resistance to DRV, using a mixture of 11 highly multi-PI-resistant HIV-1 isolates as a starting HIV-1 population. GRL-057-14 showed the most potent anti-HIV-1 activity and greatest thermal stability with wild-type protease, and potently inhibited HIV-1 protease's proteolytic activity (Ki value, 0.10 nM) among the three PIs. Structural models indicate that the C-5-isopropylamino-bis-THF moiety of GRL-057-14 forms additional polar interactions with the active site of HIV-1 protease. Moreover, GRL-057-14's P1-bis-fluoro-methylbenzene forms strong hydrogen bonding and effective van der Waals interactions. The present data suggest that the combination of C-5-aminoalkyl-bis-THF, P1-bis-fluoro-methylbenzene, and P2'-Cp-Abt confers highly potent activity against wild-type and multi-PI-resistant HIV strains and warrant further development of the three PIs, in particular, that of GRL-057-14, as potential therapeutic for HIV-1 infection and AIDS.

A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures.

BACKGROUND: Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. METHODS: To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. RESULTS: Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. CONCLUSIONS: We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

GRL-079, a Novel HIV-1 Protease Inhibitor, Is Extremely Potent against Multidrug-Resistant HIV-1 Variants and Has a High Genetic Barrier against the Emergence of Resistant Variants.

We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC50s) were 2.5 to 30 nM against wild-type HIV-1NL4-3, 0.3 to 6.7 nM against HIV-2EHO, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIVDRVrp51), with EC50s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC50s of >1,000 nM) against HIVDRVrp51 Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2' Cp-Abt group forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV11MIX failed to replicate at >0.08 µM and >0.2 µM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2' Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3 and a wide spectrum of HIVMDR strains.

A Screening Approach for the Discovery of Mechanochromic Gold(I) Isocyanide Complexes with Crystal-to-Crystal Phase Transitions.

Mechanoinduced phase transitions of emissive organic crystalline materials have received much attention. Although a variety of such luminescent mechanochromic compounds have been reported, it is challenging to develop mechanochromic compounds with crystal-to-crystal phase transitions in which precise structural information about molecular arrangements can be obtained. Here, we report a screening approach to explore mechanochromic compounds exhibiting a crystal-to-crystal phase transition. We prepared 48 para-substituted (R(1)) phenyl[para-substituted (R(2)) phenyl isocyanide]gold(I) complexes designated R(1)-R(2) (six R(1) and eight R(2) substituents) and then performed three-step screening experiments. The first screening step was selection of emissive complexes under UV light, which gave 37 emissive R(1)-R(2) complexes. The second screening step involved evaluation of the mechanochromic properties by emission spectroscopy. Twenty-eight complexes were found to be mechanochromic. The third screening step involved preparation of single crystals, reprecipitated powders, and ground powders of the 28 mechanochromic R(1)-R(2) complexes. The changes in the powder diffraction patterns of these complexes induced by mechanical stimulation were investigated. Two compounds exhibited a crystal-to-crystal phase transition upon mechanical stimulation, including the previously reported H-H complex. Single crystals of the as-prepared and ground forms of the newly discovered CF3-CN complex were obtained. Density functional theory calculations indicated that the mechanoinduced red-shifted emission of CF3-CN is caused by formation of aurophilic interactions. Comparison of the crystal structures of CF3-CN with those of the other complexes suggests that the weaker intermolecular interactions in the as-prepared form are an important structural factor for the observed mechanoinduced crystal-to-crystal phase transition.

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo.

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir.

UNLABELLED: We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 µM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE: Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) .

4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus.

UNLABELLED: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 µM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. CONCLUSION: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.

Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases, which result in a high mortality ratio and neurological sequelae after recovery. Understanding the mechanism of neuropathogenicity in these viral infections is important for the development of specific antiviral therapy. Here, we focused on human-derived neuronal and glial cells to understand the cellular responses against WNV and JEV infection. It was demonstrated that early IFN-ß induction regulated virus replication in glioblastoma tbl98G cells, whereas delayed IFN-ß induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results, which showed different IFN responses between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms for neuropathology in these viral infections.