Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants.

Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from humans to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, to evaluate the impact of S mutations, we tested 27 pseudoviruses of SARS-CoV-2 carrying different spike mutants by infecting Hela cells expressing different angiotensin-converting enzyme 2 (ACE2) orthologs from 20 animals. Of these 27 pseudoviruses, 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (B.1.429), and Mu (B.1.621). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammal ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to the spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication.

M2 of influenza virus functions as a proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularization of membranes can be performed by a variety of amphiphilic peptides, we used reverse genetics to investigate whether the peptides can substitute for M2's helix. Virus could not be generated if M2's helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor from the Epsin N-terminal homology domain or the nonnatural membrane inducer RW16. Transmission electron microscopy of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles, and ion channel activity. The protein composition and specific infectivity were also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication but that other helices can replace its basic (curvature-inducing) function.IMPORTANCE Influenza virus is unique among enveloped viruses since it does not rely on the cellular ESCRT machinery for budding. Instead, viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site, where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 that explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity.

Hemagglutinin (HA) of influenza virus is incorporated into cholesterol-enriched nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. We previously reported that mutations in the CCM retarded intracellular transport of HA and decreased its nanodomain association. Here, we analyzed whether cholesterol interacts with the CCM. Incorporation of photocholesterol into HA was significantly reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA and when HA is embedded in the membrane. We next used reverse genetics to investigate the significance of the CCM for virus replication. No virus was rescued if the whole motif is exchanged (YKLW4A); singly (LA) or doubly (YK2A and LW2A) mutated virus showed decreased titers and a comparative fitness disadvantage. In polarized cells, transport of HA mutants to the apical membrane was not disturbed. Reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses exhibit a decrease in hemolysis, which is only partially corrected if the membrane is replenished with cholesterol. More specifically, viruses have a defect in hemifusion, as demonstrated by fluorescence dequenching. Cells expressing HA YKLW4A fuse with erythrocytes, but the number of events is reduced. Even after acidification unfused erythrocytes remain cell bound, a phenomenon not observed with wild-type HA. We conclude that cholesterol binding to a group 2 HA is essential for virus replication. It has pleiotropic effects on virus assembly and membrane fusion, mainly on lipid mixing and possibly a preceding step.IMPORTANCE The glycoprotein HA is a major pathogenicity factor of influenza viruses. Whereas the structure and function of HA's ectodomain is known in great detail, similar data for the membrane-anchoring part of the protein are missing. Here, we demonstrate that the transmembrane region of a group 2 HA interacts with cholesterol, the major lipid of the plasma membrane and the defining element of the viral budding site nanodomains of the plasma membrane. The cholesterol binding motif is essential for virus replication. Its partial removal affects various steps of the viral life cycle, such as assembly of new virus particles and their subsequent cell entry via membrane fusion. A cholesterol binding pocket in group 2 HAs might be a promising target for a small lipophilic drug that inactivates the virus.

Sensitivity of transmitted and founder human immunodeficiency virus type 1 envelopes to carbohydrate-binding agents griffithsin, cyanovirin-N and Galanthus nivalis agglutinin.

Human immunodeficiency virus type 1 (HIV-1) transmission often results from infection by a single transmitted/founder (T/F) virus. Here, we investigated the sensitivity of T/F HIV-1 envelope glycoproteins (Envs) to microbicide candidate carbohydrate-binding agents (CBAs) griffithsin (GRFT), cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA), showing that T/F Envs demonstrated different sensitivity to CBAs, with IC50 values ranging from 0.006 ± 0.0003 to >10 nM for GRFT, from 0.6 ± 0.2 to 28.9 ± 2.9 nM for CV-N and from 1.3 ± 0.2 to >500 nM for GNA. We further revealed that deglycosylation at position 295 or 448 decreased the sensitivity of T/F Env to GRFT, and at 339 to both CV-N and GNA. Mutation of all the three glcyans rendered a CBA-sensitive T/F Env largely resistant to GRFT, indicating that the sensitivity of T/F Env to GRFT is mainly determined by glycans at 295, 339 and 448. Our study identified specific T/F Env residues associated with CBA sensitivity.

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.

PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness.

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

The spike receptor-binding motif G496S substitution determines the replication fitness of SARS-CoV-2 Omicron sublineage.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol.

Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a "clickable" photocholesterol compound, which closely mimics authentic cholesterol. It contains a reactive diazirine group that can be activated by UV-illumination to form a covalent bond with amino acids in its vicinity. Incorporation of photocholesterol into HA is then visualized by "clicking" it to a fluorophore, which can be detected in an SDS-gel by fluorescence scanning. This method provides a convenient and practical way to demonstrate cholesterol-binding to other proteins and probably to identify the binding site.

DC-SIGN as an attachment factor mediates Japanese encephalitis virus infection of human dendritic cells via interaction with a single high-mannose residue of viral E glycoprotein.

The skin-resident dendritic cells (DCs) are thought to be the first defender to encounter incoming viruses and likely play a role in Japanese encephalitis virus (JEV) early infection. In the current study, following the demonstration of JEV productive infection in DCs, we revealed that the interaction between JEV envelope glycoprotein (E glycoprotein) and DC-SIGN was important for such infection as evidenced by antibody neutralization and siRNA knockdown experiments. Moreover, the high-mannose N-linked glycan at N154 of E glycoprotein was shown to be crucial for JEV binding to DC-SIGN and subsequent internalization, while mutation of DC-SIGN internalization motif did not affect JEV uptake and internalization. These data together suggest that DC-SIGN functions as an attachment factor rather than an entry receptor for JEV. Our findings highlight the potential significance of DC-SIGN in JEV early infection, providing a basis for further understanding how JEV exploits DC-SIGN to gain access to dendritic cells.

Tetherin restricts HSV-2 release and is counteracted by multiple viral glycoproteins.

Tetherin has been defined as a restriction factor of HIV-1 and several other enveloped viruses. However, the significance of tetherin in viral infection remains to be further addressed. Here, we investigated whether tetherin plays a role in HSV-2 infection. Our study revealed that overexpression of tetherin restricted the release of HSV-2 into the extracellular medium, while knockdown of tetherin by siRNA enhanced its release. We further demonstrated that HSV-2 infection and viral glycoproteins gB, gD, gH and gL but not gM significantly downregulated the endogenous expression of tetherin. Additional study indicated that tetherin likely physically interacted with gB, gD, gH and gL. This is the first time that tetherin has been shown to be counteracted by multiple viral components of a virus. Our findings inform the complexity of HSV-2-host interactions, providing basis for understanding the role of tetherin as a viral restriction factor and the mechanisms underlying viral countermeasures.