The Molecular Basis for Erns Dimerization in Classical Swine Fever Virus

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein Erns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant Erns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form Erns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of Erns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

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P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for Virulence in Pigs

Journal of Virology ◽

10.1128/jvi.01104-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Libao Xie ◽

Yuying Han ◽

Yuteng Ma ◽

Mengqi Yuan ◽

Weike Li ◽

...

Keyword(s):

Viral Entry ◽

Classical Swine Fever Virus ◽

Molecular Basis ◽

Classical Swine Fever ◽

Fever Virus ◽

E2 Glycoprotein ◽

Viral Adaptation ◽

Glycoprotein E2 ◽

Domain I ◽

Highly Virulent

ABSTRACT The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain’s adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197–206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation. IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

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Effect of N-glycosylation inhibition on the synthesis and processing of classical swine fever virus glycoproteins.

Acta Biochimica Polonica ◽

10.18388/abp.2007_3172 ◽

2007 ◽

Vol 54 (4) ◽

pp. 813-819 ◽

Cited By ~ 8

Author(s):

Jolanta Tyborowska ◽

Ewa Zdunek ◽

Bogusław Szewczyk

Keyword(s):

Cell Culture ◽

Classical Swine Fever Virus ◽

Insect Cells ◽

Classical Swine Fever ◽

Fever Virus ◽

Biologically Active ◽

Complete Disappearance ◽

Dimer Formation ◽

Molecular Masses ◽

Glycosylation Inhibition

Classical swine fever virus (CSFV) is often used as a surrogate model in molecular studies of the closely related hepatitis C virus. In this report we have examined the effect of the inhibition of glycosylation on the survival and maturation of CSFV. Viral glycoproteins (E(rns), E1, E2) form biologically active complexes - homo- and heterodimers, which are indispensable for viral life cycle. Those complexes are highly N-glycosylated. We studied the influence of N-glycosylation on dimer formation using E(rns) and E2 glycoproteins produced in insect cells after infection with recombinant baculoviruses. The glycoproteins were efficiently synthesized in insect cells, had similar molecular masses and formed dimers like their natural counterparts. Surprisingly, the addition of tunicamycin (an antibiotic which blocks early steps of glycosylation) to insect cell culture blocked not only dimer formation but it also led to an almost complete disappearance of E2 even in monomeric form. Tunicamycin did not exert a similar effect on the synthesis and formation of E(rns) dimers; the dimers were still formed, which suggests that E(rns) glycan chains are not necessary for dimer formation. We have also found that very low doses of tunicamycin (much lower than those used for blocking N-glycosylation) drastically reduced CSFV spread in SK6 (swine kidney) cell culture and the virus yield. These facts indicate that N-glycosylation inhibitors structurally similar to tunicamycin may be potential therapeutics for the inhibition of the spread of CSFV and related viruses.

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ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109034 ◽

2021 ◽

Vol 255 ◽

pp. 109034

Author(s):

Liang Zhang ◽

Mingxing Jin ◽

Mengzhao Song ◽

Shanchuan Liu ◽

Tao Wang ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Ns5a Protein

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Genome Variability of Classical Swine Fever Virus during the 2018–2020 Epidemic in Japan

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109128 ◽

2021 ◽

pp. 109128

Author(s):

Tatsuya Nishi ◽

Katsuhiko Fukai ◽

Tomoko Kato ◽

Kotaro Sawai ◽

Takehisa Yamamoto

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Genome Variability

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A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

Vaccines ◽

10.3390/vaccines9050418 ◽

2021 ◽

Vol 9 (5) ◽

pp. 418

Author(s):

Youngmin Park ◽

Yeonsu Oh ◽

Miaomiao Wang ◽

Llilianne Ganges ◽

José Alejandro Bohórquez ◽

...

Keyword(s):

Vertical Transmission ◽

Classical Swine Fever Virus ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Dose ◽

Classical Swine Fever ◽

Fever Virus ◽

Tissue Samples ◽

Marker Vaccine ◽

E2 Glycoprotein

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

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