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Establishment and evaluation of a murine ???3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus.

ABSTRACT: Integrin ???3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of ???3 integrin, a stable CHO-677 cell line expressing the murine ???3 heterodimer (designated as "CHO-677-m???3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits ?? and ?3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-m???3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-m???3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable ???3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of ???3 integrin, and as a cell model for FMDV research.

SUBMITTER: Zhang W

PROVIDER: S-EPMC4588011 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Establishment and evaluation of a murine ανβ3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus.

Zhang Wei W Lian Kaiqi K Yang Fan F Yang Yang Y Zhu Zhijian Z Zhu Zixiang Z Cao Weijun W Mao Ruoqing R Jin Ye Y He Jijun J Guo Jianhong J Liu Xiangtao X Zheng Haixue H

Journal of veterinary science 20150130 3

Integrin ανβ3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of ανβ3 integrin, a stable CHO-677 cell line expressing the murine ανβ3 heterodimer (designated as "CHO-677-mανβ3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits αν and β3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), r ...[more]

PMID: 25643796

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Project description:BACKGROUND: Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules ?v?1, ?v?3, ?v?6, or ?v?8 by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the ?v?6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted ?v?6 (ss?v?6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction. RESULTS: Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ss?v?6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ss?v?6 neutralization, relative ability to utilize ?v?6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for ?v?6. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ss?v?6. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ss?v?6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding. CONCLUSIONS: These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.

Project description:Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.

Dataset Information

Establishment and evaluation of a murine ???3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus.

Publications

Establishment and evaluation of a murine ανβ3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus.

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