The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 ...receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.
APOBEC3G (A3G) is a member of the cellular polynucleotide cytidine deaminases, which catalyze the deamination of cytosine (dC) to uracil (dU) in single-stranded DNA. These enzymes potently inhibit ...the replication of a variety of retroviruses and retrotransposons, including HIV-1. A3G is incorporated into
-deficient HIV-1 virions and targets viral reverse transcripts, particularly minus-stranded DNA products, in newly infected cells. It is well established that the enzymatic activity of A3G is closely correlated with the potential to greatly inhibit HIV-1 replication in the absence of Vif. However, the details of the underlying molecular mechanisms are not fully understood. One potential mechanism of A3G antiviral activity is that the A3G-dependent deamination may trigger degradation of the dU-containing reverse transcripts by cellular uracil DNA glycosylases (UDGs). More recently, another mechanism has been suggested, in which the virion-incorporated A3G generates lethal levels of the G-to-A hypermutation in the viral DNA genome, thus potentially driving the viruses into "error catastrophe" mode. In this mini review article, we summarize the deaminase-dependent and deaminase-independent molecular mechanisms of A3G and discuss how A3G-mediated deamination is linked to antiviral mechanisms.
The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a ...critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.
ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental ...characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.
A non-filter virus inactivation unit was developed that can control the irradiation dose of aerosolized viruses by controlling the lighting pattern of a 280 nm deep-UV (DUV)-LED and the air flowrate. ...In this study, the inactivation properties of aerosolized SARS-CoV-2 were quantitatively evaluated by controlling the irradiation dose to the virus inside the inactivation unit. The RNA concentration of SARS-CoV-2 remained constant when the total irradiation dose of DUV irradiation to the virus exceeded 16.5 mJ/cm2. This observation suggests that RNA damage may occur in regions below the detection threshold of RT-qPCR assay. However, when the total irradiation dose was less than 16.5 mJ/cm2, the RNA concentration monotonically increased with a decreasing LED irradiation dose. However, the nucleocapsid protein concentration of SARS-CoV-2 was not predominantly dependent on the LED irradiation dose. The plaque assay showed that 99.16% of the virus was inactivated at 8.1 mJ/cm2 of irradiation, and no virus was detected at 12.2 mJ/cm2 of irradiation, resulting in a 99.89% virus inactivation rate. Thus, an irradiation dose of 23% of the maximal irradiation capacity of the virus inactivation unit can activate more than 99% of SARS-CoV-2. These findings are expected to enhance versatility in various applications. The downsizing achieved in our study renders the technology apt for installation in narrow spaces, while the enhanced flowrates establish its viability for implementation in larger facilities.
In HIV-1-infected patients, antiretroviral therapy (ART) is a key factor that may impact commensal microbiota and cause the emergence of side effects. However, it is not fully understood how ...long-term ART regimens have diverse impacts on the microbial compositions over time. Here, we performed 16S ribosomal RNA gene sequencing of the fecal and salivary microbiomes in patients under different long-term ART. We found that ART, especially conventional nucleotide/nucleoside reverse transcriptase inhibitor (NRTI)-based ART, has remarkable impacts on fecal microbial diversity: decreased α-diversity and increased ß-diversity over time. In contrast, dynamic diversity changes in the salivary microbiome were not observed. Comparative analysis of bacterial genus compositions showed a propensity for Prevotella-enriched and Bacteroides-poor gut microbiotas in patients with ART over time. In addition, we observed a gradual reduction in Bacteroides but drastic increases in Succinivibrio and/or Megasphaera under conventional ART. These results suggest that ART, especially NRTI-based ART, has more suppressive impacts on microbiota composition and diversity in the gut than in the mouth, which potentially causes intestinal dysbiosis in patients. Therefore, NRTI-sparing ART, especially integrase strand transfer inhibitor (INSTI)- and/or non-nucleotide reverse transcriptase inhibitor (NNRTI)-containing regimens, might alleviate the burden of intestinal dysbiosis in HIV-1-infected patients under long-term ART.
A general-purpose virus inactivation unit that can inactivate viruses was developed using deep ultraviolet (DUV) LEDs that emit DUV rays with a wavelength of 280 nm. The inside of the virus ...inactivation unit is a rectangular conduit with a sharp turn of 180° (sharp-turned rectangular conduit). Virus inactivation is attempted by directly irradiating the air passing through the conduit with DUV rays. The flow characteristics of air and virus particles inside the virus inactivation unit were investigated using numerical simulations. The air was locally accelerated at the sharp turn parts and flowed along the partition plate in the sharp-turned rectangular conduit. The aerosol particles moving in the sharp-turned rectangular conduit were greatly bent in orbit at the sharp turn parts, and then rapidly approached the partition plate at the lower part of the conduit. Consequently, many particles collided with the partition plates behind the sharp-turn parts. SARS-CoV-2 virus was nebulized in the virus inactivation unit, and the RNA concentration and virus inactivation rate with and without the emission of DUV-LEDs were measured in the experiment. The concentration of SARS-CoV-2 RNA was reduced to 60% through DUV-LED irradiation. In addition, SARS-CoV-2 passing through the virus inactivation unit was inactivated below the detection limit by the emission of DUV-LEDs. The virus inactivation rate and the value of the detection limit corresponded to 99.38% and 35.36 TCID50/mL, respectively.
Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how ...amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity.
In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.
A desktop-type air curtain system (DACS) capable of being installed on a desk to protect healthcare workers from infectious diseases was developed. Pseudo-exhaled air containing aerosol particles ...emitted from a mannequin was blown toward the air curtain generated by the DACS. The aerosol blocking effect of the DACS was investigated using particle image velocimetry measurements. A scenario in which the arm of a patient in the blood collection room is placed on the gate of the DACS was also investigated. Air curtain flow was maintained inside the gate of the DACS. The aerosol particles approaching the DACS were observed to bend abruptly toward the suction port without passing through the gate, signifying that the aerosol particles were blocked by the air curtain flow. When the arm of the patient was placed on the gate of the DACS during blood collection, the airflow above the arm was disrupted. However, the aerosol blocking performance remained unaffected. We envisage that this system will be useful as an indirect barrier not only in the medical field but also in situations where sufficient physical distance cannot be maintained, such as at the reception counter.
The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, referred to as A3) proteins are cellular cytidine deaminases that potently restrict retrovirus replication. ...However, HIV-1 viral infectivity factor (Vif) counteracts the antiviral activity of most A3 proteins by targeting them for proteasomal degradation. To date, the structure of an A3 protein containing a Vif-binding interface has not been solved. Here, we report a high-resolution crystal structure of APOBEC3C and identify the HIV-1 Vif-interaction interface. Extensive structure-guided mutagenesis revealed the role of a shallow cavity composed of hydrophobic or negatively charged residues between the α2 and α3 helices. This region is distant from the DPD motif (residues 128-130) of APOBEC3G that participates in HIV-1 Vif interaction. These findings provide insight into Vif-A3 interactions and could lead to the development of new pharmacologic anti-HIV-1 compounds.
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