SARS-CoV-2 Delta Variant Displays Moderate Resistance to Neutralizing Antibodies and Spike Protein Properties of Higher Soluble ACE2 Sensitivity, Enhanced Cleavage and Fusogenic Activity

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants’ spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

Cellular CARD11 Inhibits the Fusogenic Activity of Newcastle Disease Virus via CBM Signalosome-Mediated Furin Reduction in Chicken Fibroblasts

Newcastle disease virus (NDV) causes an infectious disease that poses a major threat to poultry health. Our previous study identified a chicken brain-specific caspase recruitment domain-containing protein 11 (CARD11) that was upregulated in chicken neurons and inhibited NDV replication. This raises the question of whether CARD11 plays a role in inhibiting viruses in non-neural cells. Here, chicken fibroblasts were used as a non-neural cell model to investigate the role. CARD11 expression was not significantly upregulated by either velogenic or lentogenic NDV infection in chicken fibroblasts. Viral replication was decreased in DF-1 cells stably overexpressing CARD11, while viral growth was significantly increased in the CARD11-knockdown DF-1 cell line. Moreover, CARD11 colocalized with the viral P protein and aggregated around the fibroblast nucleus, suggesting that an interaction existed between CARD11 and the viral P protein; this interaction was further examined by suppressing viral RNA polymerase activity by using a minigenome assay. Viral replication was inhibited by CARD11 in fibroblasts, and this result was consistent with our previous report in chicken neurons. Importantly, CARD11 was observed to reduce the syncytia induced by either velogenic virus infection or viral haemagglutinin-neuraminidase (HN) and F cotransfection in fibroblasts. We found that CARD11 inhibited the expression of the host protease furin, which is essential for cleavage of the viral F protein to trigger fusogenic activity. Furthermore, the CARD11-Bcl10-MALT1 (CBM) signalosome was found to suppress furin expression, which resulted in a reduction in the cleavage efficiency of the viral F protein to further inhibit viral syncytia. Taken together, our findings mainly demonstrated a novel CARD11 inhibitory mechanism for viral fusogenic activity in chicken fibroblasts, and this mechanism explains the antiviral roles of this molecule in NDV pathogenesis.

Heparan sulfate 3-O-sulfotransferase 4 is genetically associated with herpes zoster and enhances varicella-zoster virus–mediated fusogenic activity

Acute pain that is associated with herpes zoster (HZ) can become long-lasting neuropathic pain, known as chronic post-herpetic neuralgia (PHN), especially in the elderly. HZ is caused by the reactivation of latent varicella-zoster virus (VZV), whereas PHN is not attributed to ongoing viral replication. Although VZV infection reportedly induces neuronal cell fusion in humans, the pathogenesis of PHN is not fully understood. A genome-wide association study (GWAS) revealed significant associations between PHN and the rs12596324 single-nucleotide polymorphism (SNP) of the heparan sulfate 3- O-sulfotransferase 4 ( HS3ST4) gene in a previous study. To further examine whether this SNP is associated with both PHN and VZV reactivation, associations between rs12596324 and a history of HZ were statistically analyzed using GWAS data. HZ was significantly associated with the rs12596324 SNP of HS3ST4, indicating that HS3ST4 is related to viral replication. We investigated the influence of HS3ST4 expression on VZV infection in cultured cells. Fusogenic activity after VZV infection was enhanced in cells with HS3ST4 expression by microscopy. To quantitatively evaluate the fusogenic activity, we applied cytotoxicity assay and revealed that HS3ST4 expression enhanced cytotoxicity after VZV infection. Expression of the VZV glycoproteins gB, gH, and gL significantly increased cytotoxicity in cells with HS3ST4 expression by cytotoxicity assay, consistent with the fusogenic activity as visualized by fluorescence microscopy. HS3ST4 had little influence on viral genome replication, revealed by quantitative real-time polymerase chain reaction. These results suggest that HS3ST4 enhances cytotoxicity including fusogenic activity in the presence of VZV glycoproteins without enhancing viral genome replication.

Transmembrane Domain Dissociation Is Required for Hendra Virus F Protein Fusogenic Activity

ABSTRACT Hendra virus (HeV) is a zoonotic paramyxovirus that utilizes a trimeric fusion (F) protein within its lipid bilayer to mediate membrane merger with a cell membrane for entry. Previous HeV F studies showed that transmembrane domain (TMD) interactions are important for stabilizing the prefusion conformation of the protein prior to triggering. Thus, the current model for HeV F fusion suggests that modulation of TMD interactions is critical for initiation and completion of conformational changes that drive membrane fusion. HeV F constructs (T483C/V484C, V484C/N485C, and N485C/P486C) were generated with double cysteine substitutions near the N-terminal region of the TMD to study the effect of altered flexibility in this region. Oligomeric analysis showed that the double cysteine substitutions successfully promoted intersubunit disulfide bond formation in HeV F. Subsequent fusion assays indicated that the introduction of disulfide bonds in the mutants prohibited fusion events. Further testing confirmed that T483C/V484C and V484C/N485C were expressed at the cell surface at levels that would allow for fusion. Attempts to restore fusion with a reducing agent were unsuccessful, suggesting that the introduced disulfide bonds were likely buried in the membrane. Conformational analysis showed that T483C/V484C and V484C/N485C were able to bind a prefusion conformation-specific antibody prior to cell disruption, indicating that the introduced disulfide bonds did not significantly affect protein folding. This study is the first to report that TMD dissociation is required for HeV F fusogenic activity and strengthens our model for HeV fusion. IMPORTANCE The paramyxovirus Hendra virus (HeV) causes severe respiratory illness and encephalitis in humans. To develop therapeutics for HeV and related viral infections, further studies are needed to understand the mechanisms underlying paramyxovirus fusion events. Knowledge gained in studies of the HeV fusion (F) protein may be applicable to a broad span of enveloped viruses. In this study, we demonstrate that disulfide bonds introduced between the HeV F transmembrane domains (TMDs) block fusion. Depending on the location of these disulfide bonds, HeV F can still fold properly and bind a prefusion conformation-specific antibody prior to cell disruption. These findings support our current model for HeV membrane fusion and expand our knowledge of the TMD and its role in HeV F stability and fusion promotion.