The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163

Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3 ؉ T lymphocytes and CD14 ؉ monocytes. The VB strain primarily infects CD14 ؉ monocytes and a small subpopulation of CD3 ؉ T lymphocytes (predominantly CD4 ؉ T lymphocytes), as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has a significantly reduced ability to infect CD14؉ monocytes and has lost its capability to infect CD3 ؉ T lymphocytes. Using a panel of five recombinant chimeric viruses, we demonstrated that interactions among the GP2, GP3, GP4, GP5, and M envelope proteins play a major role in determining the CD14 ؉ monocyte tropism while the tropism for CD3 ؉ T lymphocytes is determined by the GP2, GP4, GP5, and M envelope proteins but not the GP3 protein. The data clearly suggest that there are intricate interactions among these envelope proteins that affect the binding of EAV to different cell receptors on CD3؉ T lymphocytes and CD14؉ monocytes. This study shows, for the first time, that CD3 ؉ T lymphocytes may play an important role in the pathogenesis of equine viral arteritis when horses are infected with the virulent strains of EAV.

Section: Discussionsupporting

confidence: 78%

Complex Interactions between the Major and Minor Envelope Proteins of Equine Arteritis Virus Determine Its Tropism for Equine CD3⁺T Lymphocytes and CD14⁺Monocytes

Zhang

Timoney

et al. 2010

“…Monolayer cultures of HeLa and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and the antibiotics penicillin (100 units/ml), kanamycin (20 units/ml), and streptomycin (20 units/ml) (PKS). Baby hamster kidney (BHK-21) cells were maintained as described earlier (16). The NPeGFPL stable cell line (47) derived from 293 cells was maintained as described before (47) in the presence of 1 mg/ml G418.…”

Section: Methodsmentioning

confidence: 99%

Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

Dinh

Beura

Panda³

et al. 2011

Self Cite

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections. Vesicular stomatitis virus (VSV)is an enveloped RNA virus in the family Rhabdoviridae. The negative-sense RNA genome of VSV is 11,161 nucleotides in length and encodes 5 proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix (M), the glycoprotein (G), and the large protein (L). Inside the virion, the viral genome is tightly encapsidated with the N protein to form the ribonucleocapsid complex (RNP or NC) with which the viral RNA-dependent RNA polymerase (RdRp) complex of P-L proteins (41) is associated. The G protein forms spikes on the surface of the VSV virion and mediates virus attachment as well as entry by clathrin-mediated endocytosis. Based on the low-pH environment of the endosome, the VSV G protein mediates fusion of the viral envelope with the endosomal membrane, releasing the viral RNP into the cytoplasm of the infected cell. Once inside the cytosol, the RNP undergoes primary transcription by the associated RdRp activity of the L protein along with its cofactor P protein. Translation of the transcripts by the cellular translation machinery generates viral proteins, which are used for further steps in the infection process, including genome replication, secondary transcription, assembly, and budding (53).The viral P is a multifunctional protein that modulates the association of viral replicative proteins and is indispensable for viral growth. It is required for viral genome transcription and replication (18) and also pl...

“…To our knowledge, this is the first time these results have been reported in the literature. Specifically, previous studies demonstrated that the minor envelope glycoproteins GP2 and GP4 of PRRSV interacted with pCD163 (51,52). The glycosylation of the GP2 and GP4 proteins was also shown to be required for efficient PRRSV interaction with the receptor (53,54).…”

Section: Pcd163 Srcr5 Structure For Prrsv Infectionmentioning

confidence: 99%

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

Jiang

Qiao

et al. 2017

Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus.IMPORTANCE PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.KEYWORDS CD163, Drosophila S2 cells, PRRSV, SRCR5, crystal structure P orcine reproductive and respiratory syndrome (PRRS) was first reported in 1987 in the United States (1) and has become a critical economic factor in the swine industry since its worldwide spread in the 1990s (2, 3). PRRS is characterized by