Intra- and Intermolecular Disulfide Bonds of theGP_2bGlycoprotein of Equine Arteritis Virus: Relevance forVirus Assembly andInfectivity

Abstract

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP 5 (previously named G L ) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP 2b (previously named G S ), GP 3 , and GP 4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP 5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP 2b , GP 3 , and GP 4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP 2b protein in the assembly of the GP 2b /GP 3 /GP 4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP 2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP 2b forms an intermolecular cystine bridge with one of the cysteines of the GP 4 protein, while the cysteine residues at positions 48 and 137 of GP 2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP 4 protein is responsible for the covalent association of GP 3 with the disulfide-linked GP 2b /GP 4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus which belongs to the genus Arterivirus. Other members of this single genus in the family Arteriviridae are Lactate dehydrogenase-elevating virus (LDV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Simian hemorrhagic fever virus. On the basis of conserved amino acid sequence motifs in their polymerase (poly)proteins and similarities in their genome organization and gene expression strategy, the Arteriviridae have been grouped together with the Coronaviridae and the recently established family Roniviridae in the order Nidovirales (2, 3, 4).The genome of EAV contains nine functional open reading frames (ORFs). ORF1a and -1b occupy the 5Ј three-fourths of the genome and code for proteins involved in viral replication and transcription (6,25,29,30,32). The remaining one-fourth of the viral genome contains seven relatively small ORFs (ORF2a, -2b, and -3 through -7), which are expressed from a 3Ј-coterminal nested set of six leader-containing subgenomic mRNAs (RNA2 through -7) (8, 26) and code for the known viral structur...

Envelope Protein Requirements for the Assembly of Infectious Virions of Porcine Reproductive and Respiratory Syndrome Virus

“…Here, the occurrence of a covalently linked GP 2b /GP 3 /GP 4 complex in virions could be directly demonstrated. Interestingly, while the disulfide linkage between the EAV GP 2b and GP 4 proteins appeared to be formed intracellularly, the covalent association of GP 3 , presumably by linking to GP 4 (46), occurs after the assembled virus has been released from the infected cell (45). Whether or not the proteins in the PRRSV complex are also covalently linked is presently unclear.…”

“…Whether or not the proteins in the PRRSV complex are also covalently linked is presently unclear. In this respect, it is of note that the cysteine residue (Cys 102 ), which in GP 2b of EAV is supposedly responsible for the formation of the cystine bridge with GP 4 (46), is not conserved in the GP 2a protein of PRRSV (10).…”

Sequence analysis of minor protein genes of equine arteritis virus during persistent infection

“…It has been described as an integral class I membrane protein that remains in strong association with other EAV minor glycoproteins (24). As the formation of all multimeric forms of GP2 is highly dependent on the presence of 4 Cys residues at the N-terminal end of this protein, variability at these positions is limited.…”

“…Nevertheless, their presence is essential for the production of infectious viral particles (24). It means that any changes in the amino acid sequences of the minor glycoproteins could have a significant impact on the properties of the virus.…”

İran’da Atların Viral Arteritisi Enfeksiyonunun Mevsimsel, Coğrafi, Yaş ve Cins Yayılımı

“…It may also result in abortion of pregnant mares and interstitial pneumonia or pneumoenteritis in young foals. Also, stallions can become persistently infected and shed virus into their semen [4,5] .…”