(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



VP2 residue N142 of coxsackievirus A10 is critical for the interaction with KREMEN1 receptor and neutralizing antibodies and the pathogenicity in mice [1]

['Xue Li', 'Shanghai Institute Of Infectious Disease', 'Biosecurity', 'Fudan University', 'Shanghai', 'Shanghai Institute Of Immunity', 'Infection', 'Chinese Academy Of Sciences', 'University Of Chinese Academy Of Sciences', 'Zeyu Liu']

Date: 2023-10

Coxsackievirus A10 (CVA10) has recently emerged as one of the major causative agents of hand, foot, and mouth disease. CVA10 may also cause a variety of complications. No approved vaccine or drug is currently available for CVA10. The residues of CVA10 critical for viral attachment, infectivity and in vivo pathogenicity have not been identified by experiment. Here, we report the identification of CVA10 residues important for binding to cellular receptor KREMEN1. We identified VP2 N142 as a key receptor-binding residue by screening of CVA10 mutants resistant to neutralization by soluble KREMEN1 protein. The receptor-binding residue N142 is exposed on the canyon rim but highly conserved in all naturally occurring CVA10 strains, which provides a counterexample to the canyon hypothesis. Residue N142 when mutated drastically reduced receptor-binding activity, resulting in decreased viral attachment and infection in cell culture. More importantly, residue N142 when mutated reduced viral replication in limb muscle and spinal cord of infected mice, leading to lower mortality and less severe clinical symptoms. Additionally, residue N142 when mutated could decrease viral binding affinity to anti-CVA10 polyclonal antibodies and a neutralizing monoclonal antibody and render CVA10 resistant to neutralization by the anti-CVA10 antibodies. Overall, our study highlights the essential role of VP2 residue N142 of CVA10 in the interactions with KREMEN1 receptor and neutralizing antibodies and viral virulence in mice, facilitating the understanding of the molecular mechanisms of CVA10 infection and immunity. Our study also provides important information for rational development of antibody-based treatment and vaccines against CVA10 infection.

Coxsackievirus A10 (CVA10) is an emerging pathogen that causes hand, foot, and mouth disease. No approved vaccines or antiviral treatments for CVA10 infection are yet available. Here we identified VP2 N142 of CVA10 as a key residue essential for viral recognition by KREMEN1 receptor by selection of soluble receptor neutralization-resistant mutant and mutational analysis. Residue N142 when mutated could significantly reduce receptor-binding activity, viral attachment, and infection in vitro. Mouse infection experiments show that residue N142 when mutated could reduce fatality rate and severity of symptoms by decreasing viral loads in limb muscle and spinal cord. Furthermore, residue N142 when mutated could confer resistance to neutralization by anti-CVA10 neutralizing antibodies. Additionally, N142, which is exposed on the canyon rim but highly conserved in CVA10, is identified as a key receptor-binding residue, providing a counterexample to the canyon hypothesis. Overall, our findings promote a better understanding of the molecular basis of CVA10 attachment and infection and in vivo pathogenicity, and also provide useful information for rational research and development of vaccines and antibody-based treatment against CVA10 infection.

Funding: C.Z. is supported by the Shanghai Municipal Science and Technology Major Project (ZD2021CY001) and Shanghai Rising-Star Program (21QA1410000). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Key residues in the CVA10 capsid proteins that are critical for viral binding to KRM1 and viral infection have not been identified by experiment, such as mutational analysis. In this study, we identified VP2 N142 as a key residue responsible for CVA10 interaction with KRM1 receptor by screening of CVA10 mutants that were resistant to neutralization by soluble KRM1 protein. N142 is located at the tip of VP2 EF loop and exposed on the virion surface. We demonstrated that residue N142 when mutated drastically reduced CVA10 attachment and infection in cell culture by reducing virus binding to KRM1 receptor. More importantly, mouse infection experiments showed that residue N142 when mutated could reduce fatality rate and severity of symptoms by decreasing viral loads in limb muscle and spinal cord, indicating the importance of N142 for the pathogenicity of CVA10 in vivo. Furthermore, residue N142 was shown to be a critical part of the epitopes recognized by anti-CVA10 polyclonal antibodies and a neutralizing monoclonal antibody (MAb) called 2A11. N142 when mutated could confer resistance to neutralization by the anti-CVA10 neutralizing antibodies. Overall, our study demonstrates that VP2 residue N142 of CVA10 is a key residue that plays an important role in the interactions with KRM1 receptor and neutralizing antibodies and viral virulence in mice.

CVA10 (prototype strain Kowalik) was first isolated in the United States in 1950 [ 22 ]. It belongs to species A of the genus Enterovirus in the family Picornaviridae [ 23 ]. Like other enteroviruses, CVA10 is a non-enveloped, single-stranded RNA virus and has an icosahedral capsid comprised of 60 copies of VP1, VP2, VP3, and internal VP4 [ 23 ]. Cryo-electron microscopy (cryo-EM) has been used to study the 3D structures of CVA10 viral particles, including mature virion, A particle (uncoating intermediate), and procapsid (empty capsid). The structures reveal that on the CVA10 virion surface, there are a star-shaped plateau on each of the 5-fold axes, a depression (termed the canyon) encircling the plateau, and a propeller-like protrusion centered on each 3-fold axis [ 24 – 28 ]. The cell surface protein KREMEN1 (KRM1) has been identified as an entry receptor for CVA10 [ 29 ]. KRM1 selectively binds to mature CVA10 virion above the canyon [ 26 , 28 ].

Coxsackievirus A10 (CVA10) is a viral pathogen that causes hand, foot and mouth disease (HFMD), which is a common infectious disease and usually affects infants and young children [ 1 ]. Moreover, CVA10 may also cause a variety of complications, including cough, bronchitis, pneumonia, diarrhea, vomit, myocarditis, cardiac damage, meningitis, encephalitis, brain myelitis, coma, acute flaccid paralysis, or seizures [ 2 , 3 ]. CVA10 infections are generally mild and self-limiting, but may be severe and life threatening in risk groups such as infants [ 3 , 4 ]. In recent years, numerous HFMD cases and outbreaks associated with CVA10 infection have been reported in China and many other countries worldwide [ 5 – 15 ], highlighting that CVA10 has become one of the major causative agents of HFMD. For now, no approved vaccine or drug is available for CVA10, although several CVA10 vaccine candidates have been tested in animal models [ 16 – 21 ].

Results

Isolation of CVA10 mutants resistant to neutralization with soluble KRM1 receptor We generated a soluble form of human KRM1 consisting of the ectodomain (residues A23 to G373) fused to the Fc portion of human IgG1, designated KRM1-Fc. The KRM1-Fc protein was able to effectively neutralize CVA10 prototype strain Kowalik with neutralization concentration (100% protection) of 12.5 nM (Fig 1A and 1B). To identify the CVA10 residues involved in KRM1 receptor binding, we attempted to isolate and characterize CVA10 mutants that escaped neutralization with KRM1-Fc (Fig 1A and 1B). CVA10/Kowalik was subjected to three passages in the presence of gradually increasing concentrations of KRM1-Fc (Fig 1A). The neutralization-resistant mutants readily emerged when treated with the appropriate concentrations of KRM1-Fc (Fig 1B). The mutants from two different wells (the upper and lower wells were designated well #1 and #2, respectively) of the culture plate were subjected to plaque purification, and the sequences of the capsid protein-coding region of these isolates were determined and aligned with that of wild-type CVA10/Kowalik. The information of the plaque-purified isolates is summarized in Fig 1B. Among the 10 isolates from well #1, 7 had double mutations: N→D at residue 142 of VP2 (N2142D) and N→Y at residue 183 of VP3 (N3183Y), and 2 had an additional mutation at position 1287 (A1287T) or 2156 (T2156S) besides the N2142D+N3183Y mutation, and the last one possessed a double mutation in the VP1 protein (T1219I and S1238G). Note that viral capsid residues are numbered from 1001, 2001, and 3001 in VP1, VP2, and VP3, respectively. Among the 9 isolates from well #2, 4 had the N3183Y substitution, and the other 4 possessed double mutations at residues 1219 and 1238 (T1219I and S1238G), and the remaining one had a double mutation (N2142D+N3183Y). Thus, there are three major types of mutations: N2142D+N3183Y, N3183Y, and T1219I+S1238G (Fig 1C), which were used for the subsequent analyses. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Selection of CVA10 mutants resistant to neutralization with soluble human KRM1 receptor. (A) A flowchart of the screening procedure. (B) Screening and information of soluble KRM1-resistant mutants. Green circle, no CPE; orange, partial CPE; red, complete CPE. 10 and 9 plaques were isolated from well #1 (the upper well) and #2 (the lower well), respectively, and then sequenced to identify mutations. Viral capsid residues are numbered from 1001, 2001, and 3001 in VP1, VP2, and VP3, respectively. (C) A summary of the three major types of the mutations. The red arrows indicate the substitutions in the first or second nucleotides of the codons. https://doi.org/10.1371/journal.ppat.1011662.g001

The mutation at residue N2142 is responsible for the soluble receptor-resistant phenotype Each of the mutants N2142D+N3183Y, N3183Y, and T1219I+S1238G was titrated by TCID 50 assay and then tested for resistance to neutralization by KRM1-Fc by standard neutralization assay. The results are summarized in Fig 2A. The titers of the mutants N3183Y and T1219I+S1238G were comparable to that of wildtype CVA10/Kowalik (CVA10-WT), while the titer of the mutant N2142D+N3183Y were 25 times lower than that of CVA10-WT, indicating that N2142D, but not the other mutations, may reduce viral fitness. Furthermore, the mutants containing the N3183Y substitution or the T1219I+S1238G mutation were sensitive to neutralization with KRM1-Fc, whereas the mutant N2142D+N3183Y was resistant to KRM1-Fc and the neutralization concentration decreased by more than 128 times compared with CVA10-WT. These results suggest that N2142D, but not the N3183Y, T1219I, and S1238G mutations, is likely to be responsible for the soluble receptor-resistant phenotype. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. The role of the mutations in conferring resistance to neutralization with soluble KRM1-Fc. (A) Wildtype and mutant CVA10 viruses were titrated by TCID 50 assay and analyzed for resistance to neutralization with KRM1-Fc. Fold increase or decrease (prefixed with a minus sign) in viral titer and neutralization concentration of KRM1-Fc against the mutants, relative to wildtype CVA10. Red highlighting, more than 10-fold decrease. (B) Schematic representation of construction of the T7 promoter (P T7 )-driven infectious clone of CVA10. The indicated mutations were separately introduced into the infectious clone. (C) Schematic of rescue and characterization of wildtype and mutant CVA10 viruses. T7 RNA-pol, T7 RNA polymerase. (D) The rescued CVA10 viruses were titrated by TCID 50 assay and sequenced. dpi, day post infection. (E) The rescued CVA10 viruses were analyzed for resistance to neutralization with KRM1-Fc. https://doi.org/10.1371/journal.ppat.1011662.g002 To further confirm the above conclusion, a T7 promoter-driven infectious clone of CVA10/Kowalik was constructed, and the mutations, N3183Y, N3183A, N2142D, T1219I, and S1238G, were separately introduced into the CVA10 infectious clone using site-directed mutagenesis (Fig 2B). The N3183A mutation was designed to change the polarity of N3183 residue from polar to non-polar. The wildtype and mutant CVA10 viruses were rescued by co-transfecting HEK 293T cells with CVA10 infectious clone plasmid and the T7 RNA polymerase-encoding plasmid and amplified in RD cells (Fig 2C). The rescued CVA10 viruses were sequenced and titrated by TCID 50 assay. The results are summarized in Fig 2D. CVA10 viruses WT, N3183Y, N3183A, T1219I, and S1238G were successfully rescued and their infectious viral titers were greater than or equal to 107 TCID 50 /ml. We failed to rescue the N2142D mutant, because reverse mutation (D→N at residue 2142) occurred in cell culture. To address this issue, we constructed a new CVA10 mutant N2142A and found that this mutant could be successfully rescued using the reverse genetics and was genetically stable, although its infectious viral titer was significantly lower than that of the rescued CVA10-WT (Fig 2B–2D). Next, each of the rescued viruses, WT, N3183Y, N3183A, N2142A, T1219I, and S1238G, was tested for resistance to neutralization by KRM1-Fc. Compared with the rescued CVA10-WT, rescued viruses N3183Y, N3183A, T1219I, and S1238G were still sensitive to neutralization with KRM1-Fc, while N2142A showed 64-fold increase of resistance to KRM1-Fc (Fig 2E). Together, these results demonstrate that the mutation at residue N2142, but not N3183, T1219, and S1238, is responsible for the soluble receptor-resistant phenotype.

The N2142A mutation could reduce viral replication and binding to cell by impairing receptor binding Growth kinetics of rescued CVA10-WT and mutant N2142A (CVA10-N142A) were compared. RD cells were infected at a multiplicity of infection (MOI) of 0.01 and viral titers were determined at 0, 6, 12, 24, 36, and 48 hours post infection by TCID 50 assay (Fig 4A). Both CVA10-WT and CVA10-N142A viruses replicated well in RD cells. There were no significant differences in viral titers at 0, 6, and 12 hours post infection between the two groups. However, CVA10-N142A grew significantly worse than CVA10-WT at 24, 36, and 48 hours post infection, and the N2142A mutation resulted in nearly a 10-fold reduction in virus titer, indicating that N2142A mutation is able to significantly reduce CVA10 replication in cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. N2142A mutation reduced viral replication and binding to cell by impairing receptor binding. (A) Growth kinetics of rescued CVA10-WT and CVA10-N142A were compared in RD cells. RD cells were infected at a MOI of 0.01 and viral titers were determined at the indicated time points post infection. Data are means ± SD of three replicate samples. Analysis was performed using two-way ANOVA. ns, no significant difference (p ≥ 0.05); **, p < 0.01; ****, p < 0.0001. (B) CVA10-WT or CVA10-N142A infected cell cultures were subjected to sucrose gradient ultracentrifugation, and the resulting 12 fractions were analyzed by SDS-PAGE. The fraction #10 samples (indicated by red arrows) were selected for subsequent analysis. (C) The fraction #10 samples of CVA10-WT and CVA10-N142A were diluted to 50 μg/ml and subjected to negative stain electron microscopy. Red and blue arrows indicate full (mature virion) and empty CVA10 particles, respectively. (D) The fraction #10 samples of CVA10-WT and CVA10-N142A were diluted to 1 μg/ml and assayed for viral titers. Fold decrease (prefixed with a minus sign) in viral titer is also shown. (E) 100 ng/ml of CVA10 viral particles (fraction #10) were attached to prechilled RD cells at 4°C for 1 h, and after washing, the levels of CVA10 RNA were determined by RT-qPCR. Data are means ± SD of four replicate samples. The Mann–Whitney test was used to compare differences. *, p < 0.05. (F) Reactivity of CVA10-WT and CVA10-N142A viral particles with human KRM1-Fc protein were determined by ELISA. CVA10 viral particles were serially diluted and coated onto microplates, followed by incubation with biotinylated KRM1-Fc or ACE2-Fc (ctr) protein and HRP-conjugated streptavidin. Data are means ± SD of three replicate samples. (G) Proposed model of residue N2142-dependent CVA10 attachment and infection of target cells. The N2142A mutation is indicated by red dots. https://doi.org/10.1371/journal.ppat.1011662.g004 To determine the influence of the N2142A mutation on the assembly of CVA10 viral particles, the supernatants of CVA10-WT or CVA10-N142A infected cell cultures were clarified, concentrated, and then subjected to sucrose gradient ultracentrifugation (Fig 4B). SDS-PAGE analysis of the resultant gradient fractions showed that the patterns of capsid protein composition and content were generally similar for the CVA10-WT and CVA10-N142A samples. Both viruses consisted of VP1, VP3, VP0 or/and VP2 proteins, and these viral proteins co-sedimented mainly in fractions #7 to #11. Note that infectious mature CVA10 virion consisted of VP1, VP3, VP2, and VP4 proteins, while non-infectious CVA10 empty particles (also termed procapsid) were composed of VP1, VP3, and VP0 proteins [25]. These results indicate that the surface-exposed N2142A mutation does not affect CVA10 viral assembly. The fraction #10 samples of CVA10-WT and CVA10-N142A were selected for subsequent analysis, because viral particles in this fraction were mainly composed of VP1, VP2, and VP3 proteins, corresponding to mature CVA10 virion. To verify formation of viral particles, the fraction #10 samples of CVA10-WT and CVA10-N142A were diluted to 50 μg/ml in PBS and subjected to negative stain electron microscopy (EM). As shown in Fig 4C, ~30 nm spherical particles were observed for both virus samples. And importantly, the vast majority of the CVA10-WT and CVA10-N142A virus particles were full particles, corresponding to mature virion. However, CVA10-N142A at the same mass concentration had lower viral titers compared to CVA10-WT. Specifically, viral titers of fraction #10 of CVA10-WT and CVA10-N142A at 1 μg/ml were determined to be 108.5 and 106.6 TCID 50 /ml, respectively, which was an 86-fold difference (Fig 4D). These results suggest that at the same infectious viral titer, CVA10-N142A has much higher particle number than CVA10-WT. Viral particles with the same mass concentration, but not the same infectious viral titer, were used to compare the differences between CVA10-WT and CVA10-N142A in terms of cell-binding capacity. 100 ng/ml of CVA10 viral particles (fraction #10) were adsorbed to cooled RD cells at 4°C for 1 h to allow cell binding but not entry, and after washing to remove unbound virus, levels of RNA of CVA10 particles bound to cells were determined by RT-qPCR (Fig 4E). Compared with CVA10-WT, cell-binding activity of CVA10-N142A decreased by 36%, and significant differences were observed between the two groups, indicating that the N2142A mutation could impair virus binding to the cell surface. We then compared CVA10-WT and CVA10-N142A for binding with soluble human KRM1-Fc protein by ELISA assay. In this assay, CVA10 viral particles (fraction #10) were serially diluted and coated onto ELISA plates, followed by incubation with biotin-labeled human KRM1-Fc protein and HRP-conjugated streptavidin (Fig 4F). CVA10-WT efficiently reacted with human KRM1-Fc in virus dose-dependent fashion, whereas CVA10-N142A reacted with KRM1-Fc only at high virus concentration (≥ 2 μg/ml) but not at low concentrations. Moreover, the KRM1-Fc-binding levels of CVA10-N142A were much lower than those of CVA10-WT. The results demonstrate that the N2142A mutation could reduce CVA10 binding affinity to KRM1 receptor, in line with the results of the cell-attachment assay (Fig 4E). We proposed a CVA10 infection model to show the importance of residue N2142 in CVA10 attachment and infection processes (Fig 4G). Compared with CVA10-WT, the N2142A mutation could impair virus binding to KRM1 receptor on the cell surface, leading to a significant decrease in the production of progeny virus.

The N2142A mutation could reduce viral pathogenicity in mice To evaluate the virulence and pathogenicity of the CVA10 viruses in vivo, groups of 2-day-old ICR mice were infected with 300 TCID 50 of rescued CVA10-WT or CVA10-N142A, and then clinical symptoms and mortality were observed daily for 14 days. As shown in Fig 5A, CVA10-WT-infected mice rapidly developed limb weakness and paralysis and had mortality rate of 92.3% (12/13). By contrast, only four out of 12 (33.3%) CVA10-N142A-inoculated mice developed limb paralysis and eventually died, and the other eight only had mild symptoms or no symptoms. There was a significant difference in the mortality rate between the two groups (p = 0.0001). The results demonstrate that the N2142A mutation can decrease the virulence of CVA10 in mice. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. N2142A mutation could reduce mortality rate and severity of symptoms by decreasing viral loads in limb muscle and spinal cord. (A) Groups of 2-day-old ICR mice were inoculated with 300 TCID 50 of CVA10-WT or CVA10-N142A, and then survival (left panel) and clinical symptoms (right panel) were observed daily for 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death. Number of mice per group were indicated in brackets. Statistical significance of survival curves between groups was determined by Log-rank (Mantel-Cox) test. ***, p < 0.001. All error bars represent SEM. (B) Groups of ICR mice infected with CVA10-WT or CVA10-N142A were sacrificed at 4 dpi, and limb muscle (left panel), spinal cord (middle panel), and brain (right panel) were collected, weighed, and tested for virus titers. There were 12 mice per group. Each symbol represents an individual mouse. The Mann–Whitney test was used to compare differences. **, p < 0.01; ***, p < 0.001. (C) Correlations between viral titers in the indicated organs and clinical scores. Viral titers and clinical scores of individual infected mice from the CVA10-WT and CVA10-N142A groups were used for Pearson correlation coefficient analysis. *, p < 0.05; **, p < 0.01. https://doi.org/10.1371/journal.ppat.1011662.g005 To determine viral loads in organs of infected mice, groups of ICR mice inoculated with CVA10-WT or CVA10-N142A were sacrificed at 4 days post infection (dpi), and limb muscle, spinal cord, and brain were harvested for determination of virus titers by TCID 50 assay (Fig 5B). Viral titers per gram of limb muscle tissues from the CVA10-WT and CVA10-N142A groups ranged from 3088 to 902,857 (geometric mean titer = 109,903) and from 4216 to 200,000 (geometric mean titer = 9768), respectively. In addition, viral titers per gram of spinal cord samples from the CVA10-WT and CVA10-N142A groups ranged from undetectable to 920,000 (geometric mean titer = 10,783) and from undetectable to 2107 (geometric mean titer = 290), respectively. There were significant differences in viral titers in limb muscle and spinal cord between the CVA10-WT and CVA10-N142A groups. Moreover, there were significant positive correlations between the viral titers in limb muscle and spinal cord and the clinical scores of infected mice (Fig 5C). However, no statistically significant differences were observed in brain viral titers between the CVA10-WT and CVA10-N142A groups (Fig 5B). Furthermore, no significant correlations between brain viral titers and clinical scores were found (Fig 5C). Taken together, the results imply that the N2142A mutation could reduce the fatality rate and severity of symptoms by decreasing viral loads in limb muscle and spinal cord but not brain. To determine histopathological changes after infection, groups of ICR mice inoculated with CVA10-WT or CVA10-N142A were sacrificed at 4 dpi, and limb muscle samples were collected and subsequently used for histopathologic examination. Hematoxylin and eosin (H&E) staining images of infected and normal limb muscle tissues are shown in Fig 6A. Note that severity of myositis was graded on a scale of 0 to 4. For the CVA10-WT group, only one out of 15 (6.7%) infected mice showed normal muscle fibers, while the other 14 mice displayed varying degrees of limb muscle tissue damage. 5 out of 11 (45.5%) CVA10-N142A-infected mice showed normal limb muscle morphology, and the other 6 mice developed necrotizing myositis. The CVA10-WT group had a significantly higher mean histopathologic score compared to the CVA10-N142A group (p = 0.0046) (Fig 6B). Therefore, CVA10-N142A-infected mice showed a milder limb muscle pathology compared to CVA10-WT-infected mice. In addition, clinical scores were found to correlate well with the degree of limb muscle tissue destruction (p = 0.0178) (Fig 6C). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. N2142A mutation could lead to milder limb muscle pathology in infected mice and significantly reduce CVA10 binding affinity to murine KRM1 receptor. (A) Groups of ICR mice inoculated with medium (control), CVA10-WT, or CVA10-N142A were sacrificed at 4 dpi, and limb muscle samples were collected for histological examination. Histopathologic scores were graded according to the severity of myositis as: not present, mild, moderate, and severe. Number of mice (n) is shown. (B) Histopathologic scores of the CVA10-WT and CVA10-N142A groups. Each symbol represents an individual mouse. The Mann–Whitney test was used to compare differences. **, p < 0.01. The solid line indicates the mean values. (C) Correlations between histopathologic scores and clinical scores. Histopathologic scores of individual mice from the CVA10-WT and CVA10-N142A groups were used for Pearson correlation analysis. *, p < 0.05. (D) Reactivity of CVA10-WT and CVA10-N142A viral particles with murine KRM1-Fc (mKrm1-Fc) were analyzed by ELISA. Serially diluted Viral particles were coated onto microplates and then incubated with biotinylated KRM1-Fc or ACE2-Fc (ctr) protein and HRP-conjugated streptavidin. Data are means ± SD of three replicate samples. (E) Sequence alignment of the human (hKRM1) and mouse (mKrm1) KRM1 proteins. The three KRM1 residues (D88, G89, and D90) predicted to contact CVA10 residue N142 are boxed. The graph was generated using CLC Sequence Viewer software. https://doi.org/10.1371/journal.ppat.1011662.g006 To explore why the N142A mutation leads to significantly lower virulence in mice, CVA10-WT and CVA10-N142A were compared for binding with soluble murine KRM1-Fc (mKrm1-Fc) protein by ELISA. mKrm1 was reported to be required for CVA10 infection in mouse, as mKrm1-deficient mice are resistant to CV-A10-induced lethal paralysis [29]. Serially diluted CVA10 viral particles were coated onto ELISA plates and then allowed to incubate with biotin-labeled mKrm1-Fc protein and HRP-conjugated streptavidin (Fig 6D). CVA10-WT could effectively react with mKrm1-Fc in virus dose-dependent manner, whereas CVA10-N142A did not show any obvious binding even at the highest virus concentration tested (4 μg/ml). Thus, the N2142A mutation could significantly reduce CVA10 binding affinity to murine KRM1 receptor. The mouse and human KRM1 proteins share 93.7% sequence identity, and the three residues (D88, G89, and D90) predicted to contact CVA10 residue N142 are identical in the two KRM1 proteins (Fig 6E). When N2142 of CVA10 is mutated to Ala, the hydrogen bond interactions with D88 and G89 of murine KRM1 would be lost, as analyzed above (Fig 3C).

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011662

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.