Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

Gladue

et al. 2011

E1, along with E rns and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E rns loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1⌬Cys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1⌬Cys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, Classical swine fever virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with Bovine viral diarrhea virus (BVDV) and Border disease virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviviridae (2). The 12.3-kb CSFV genome contains a single open reading frame that encodes a 3,898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH 2 -Npro-C-E rns -E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co-and posttranslational processing of the polyprotein by cellular and viral proteases (5). Structural components of the CSFV virion include the capsid (C) protein and glycoproteins E rns , E1, and E2. E1 and E2 are anchored to the envelope at their carboxyl termini, and E rns loosely associates with the viral envelope (22, 23). E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (19). E1 has been implicated (21), along with E rns and E2 (4), in viral adsorption to host cells. Importantly, different modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (3,6,9,10,11,12,15,20).In lysates of CSFV-infected cells, E2 glycoprotein forms homodimers and heterodimers with E1 glycoprotein (14,22) via disulfide bridges between cysteine residues. Formation of these disulfide-linked heterodimers also occurs in CSFV virions (19), suggesting that the interaction between envelope proteins plays an important role in virus ...

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Substitution of Specific Cysteine Residues in the E1 Glycoprotein of Classical Swine Fever Virus Strain Brescia Affects Formation of E1-E2 Heterodimers and Alters Virulence in Swine

Fernandez-Sainz

Gladue

et al. 2011

“…E2 has been implicated, along with E rns (14) and E1 (43), in viral adsorption to host cells; indeed, chimeric pestiviruses exhibit infectivity and cell tropism phenotypes consistent with those of the E2 gene donor (18,40). Modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (21,29,31,41).…”

mentioning

confidence: 99%

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

Risatti

Sainz

et al. 2007

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.

“…E2 has been implicated, along with E rns (8) and E1 (9), in viral adsorption to host cells (10,11). Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence (12)(13)(14)(15)(16).…”

Section: Importancementioning

confidence: 99%

Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine

Largo

Gladue

et al. 2016

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a WimleyWhite interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond ( 869 CKWGGNWTCV 878 , named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872 GG 873 dipeptide, as well as two aromatic residues ( 871 W and 875 W) accessible to solvent. Reverse genetics utilizing a fulllength infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. IMPORTANCEThis report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs. C lassical swine fever (CSF) is a highly contagious disease of swine caused by CSF virus (CSFV), a small enveloped virus with a positive-sense, single-strand RNA genome (1). The approximately 12.5-kb CSFV genome contains a single open reading frame that encodes a polyprotein composed of 3,898 amino acids that ultimately yields up to 12 final cleavage pr...