Structure and function of Gp3 of porcine reproductive and respiratory syndrome virus
Final Report Abstract
Arteriviruses, enveloped RNA viruses, have long been a challenge in veterinary medicine. This study focuses on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a crucial pathogen in the swine industry. PRRSV strains have spread worldwide and diversified rapidly since their discovery, resulting also in the emergence of highly pathogenic variants. We investigated the structure and processing of the membrane proteins, a dimer composed of glycoprotein 5 (Gp5) and M, and a trimer formed by Gp2, Gp3, and Gp4. Gp5 and M are linked to fatty acids at cysteines in the cytoplasmic domain. This modification is essential for virus replication, facilitating the assembly of viral proteins on internal membranes. An artificial intelligence-based system, AlphaFold2, provides an unprecedented insight into the unique structural conformation of Gp5 and M, which might allow these proteins to deform lipid membranes, a crucial step in the budding of virus particles. Genetic overlap between the genes encoding most membrane proteins poses challenges for genetic manipulation, making it difficult to insert tags into the viral genome, which are required for antibody detection. Our study uncovers that these inserted tags are unstable and tend to revert to their wild-type sequences after a few passages, suggesting a yet-undefined fitness advantage associated with overlapping genes. We successfully generate a virus bearing an HA-tag at the C-terminus of Gp4, which does not overlap with the next gene. We then focused on the complex composed of the small glycoproteins Gp2, Gp3 and Gp4. Gp2 and Gp4 are typical type 1 transmembrane proteins, but Gp3 has a unique hairpin-like membrane topology: Both the N- and C-terminus are exposed to the lumen of the ER and an internal amphiphilic helix peripherally anchors the protein to the membrane. It is discovered that while the C-terminus proves beneficial for PRRSV replication, it is not essential. In contrast, mutations in the amphiphilic helix tend to be either lethal or result in severe growth defects. This underscores the crucial role of the helix's biophysical properties and its specific amino acid sequence, implying a function beyond Gp3 membrane anchoring. The structure of Gp2/Gp3/Gp4 trimer predicted by alphafold2 shows that the amphiphilic helix of Gp3 is inserted into the membrane, proximate to the transmembrane region of Gp2 and Gp4. This suggests that the amphiphilic helix might be driving membrane fusion during virus entry. In contrast, the C-terminus is unstructured, in accordance with its high amino acid variability between PRRSV strains. The absence of disulfide bonds between the small glycoproteins in the predicted structure distinguishes it from the equine arteritis virus, the Arteriviridae prototype, a finding experimentally validated using the Gp4-HA virus. In summary, this project significantly improves our understanding of Arteriviruses and sheds light on their unique structural and functional properties.
Publications
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Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth. PLOS Pathogens, 17(4), e1009554.
Zhang, Minze; Han, Xiaoliang; Osterrieder, Klaus & Veit, Michael
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Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells. Viruses, 14(4), 749.
Matczuk, Anna Karolina; Zhang, Minze; Veit, Michael & Ugorski, Maciej
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Using Alphafold2 to Predict the Structure of the Gp5/M Dimer of Porcine Respiratory and Reproductive Syndrome Virus. International Journal of Molecular Sciences, 23(21), 13209.
Veit, Michael; Gadalla, Mohamed Rasheed & Zhang, Minze
