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The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein 1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals. Chronic infection with SARS-CoV-2 leads to the emergence of viral variants that show reduced susceptibility to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives’ action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription—without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives’ oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load 1 , 2 . However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir. A specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are almost exclusively found in sequences from 2022, after molnupiravir treatment was introduced, indicating that molnupiravir treatment can give rise to viable mutagenized viruses.

Molnupiravir, a wide-spectrum antiviral that is currently in phase 2/3 clinical trials for the treatment of COVID-19, is proposed to inhibit viral replication by a mechanism known as ‘lethal mutagenesis’. Two recently published studies reveal the biochemical and structural bases of how molnupiravir disrupts the fidelity of SARS-CoV-2 genome replication and prevents viral propagation by fostering error accumulation in a process referred to as ‘error catastrophe’.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene–protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication. This Review outlines the gene and protein expression strategies used by viruses to expand the efficiency of their coding and regulatory sequences, and the implications of these mechanisms for developing antiviral agents.

Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies.

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

Vaginal microbiota modulates susceptibility to sexually transmitted infections and produces carboxylic acid metabolites that have antimicrobial activity; however, their activity against viral sexually transmitted infections is not well defined. We determined the HIV-1 virucidal activity of lactic acid (LA), short chain fatty acids (SCFAs), and succinic acid, representing conditions observed in women with an optimal Lactobacillus -dominated vaginal microbiota compared to women with bacterial vaginosis. Virucidal activity against enveloped HIV-1 and HSV-2, the non-enveloped HPV16, and the mechanism by which LA inactivates HIV-1 was further assessed. LA was > 10-fold more potent at inactivating an HIV-1 transmitted/founder strain than SCFAs and succinic acid when tested at an equivalent 20 mM of protonated acid (p≤0.05). While LA decreased HIV-1 infectivity by >10 3 -fold, virions were intact, expressed a similar gp120:p24 ratio, and showed only a 2-fold decrease in CD4 binding compared to untreated HIV-1 (p≤0.05). Treatment of recombinant gp120 with LA revealed no major conformational changes by small angle X-ray scattering. LA treatment of HIV-1 resulted in an 80% decrease in virion-associated reverse transcriptase activity compared to untreated virus (p < 0.01), which was more potent than acetic acid or HCl-adjusted media at the same pH, with this effect observed in the presence of cervicovaginal fluid. LA decreased HIV-1 virion-associated RNA levels by ∼50% compared to untreated virus (p < 0.001), acetic acid or HCl acidified media. In contrast, HSV-2 virucidal activity of LA was similar to acetic acid and HCl-acidified media while HPV16 was acid-resistant. Our results demonstrate LA’s potent and specific HIV-1 virucidal activity compared to SCFAs and succinic acid found in the female reproductive tract, and its HIV-1 virucidal mechanism mediated by penetration of the viral membrane and core to target a key viral enzyme and nucleic acid. These findings have implications for the vaginal transmission of HIV to partners and neonates during birth.