Complex interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes

Abstract

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.

FIG. 1.

Differences in susceptibility of CD3⁺ T lymphocytes to infection with the VB and MLV strains of EAV. Dual-color immunofluorescence flow cytometric analysis was performed at various times postinfection. (A) Values represent the mean percentages (± the standard error of the mean) of CD3⁺ T cells infected with VB (black circles) and MLV (white circles) for six horses with T lymphocytes susceptible to EAV VB infection (group A). Percentages of infected cells at the indicated times (data points on the curve) were determined by using dot plots derived from dual-fluorescence flow cytometric analyses of cells (representative dot plots of VB infection, insets a through d; MLV infection, insets e through h). (B) Values represent the mean percentages (± the standard error of the mean) of CD3⁺ T cells infected with VB (black circles) and MLV (white circles) for four horses with T lymphocytes resistant to EAV VB infection (group B) as determined by using dot plots derived from dual-fluorescence flow cytometric analyses of cells (representative dot plots of VB infection, insets a through d; MLV infection, insets e through h).

FIG. 2.

Susceptibility differences between CD4⁺ and CD8⁺ T lymphocytes and CD21⁺ B lymphocytes to infection with the VB and MLV strains of EAV. Dual-color immunofluorescence flow cytometric analysis was performed at various times postinfection. Values represent the mean percentages (± the standard error of the mean) of (A) CD4⁺ T cells, (B) CD8⁺ T cells, and (C) CD21⁺ B cells infected with the VB (black circles) and MLV (white circles) strains of EAV. Percentages of infected cells at the indicated times (data points on the curve) were determined by using dot plots derived from dual-fluorescence flow cytometric analyses of cells (representative dot plots of VB infection, insets a through d; MLV infection, insets e through h).

FIG. 3.

Infection of monocytes with the VB and MLV strains of EAV. (A and B) Dual-color immunofluorescence flow cytometric analysis was performed at various times postinfection. Values represent the mean percentages (± the standard error of the mean) of monocytes infected with VB (black circles) and MLV (white circles) for (A) two horses with T lymphocytes susceptible to EAV VB infection (group A) and (B) two horses with T lymphocytes resistant to EAV VB infection (group B). Percentages of infected cells at the indicated times (data points on the curve) were determined by using dot plots derived from dual-fluorescence flow cytometric analyses of cells (representative dot plots of VB infection, insets a through d; MLV infection, insets e through h).

FIG. 4.

Detection of EAV N protein expression in T lymphocytes infected with the VB strain. CD3⁺, CD4⁺, and CD8⁺ T lymphocytes from group A horses infected with the VB and MLV strains of EAV were examined by dual-color immunofluorescence flow cytometric analysis using MAbs against the EAV nsp1 (MAb 12A4) and N (MAb 3E2) proteins and MAbs for cell-specific cell surface antigens at 24 hpi. (a through c) Lymphocytes infected with the VB strain were stained with anti-EAV nsp1 MAb AF488 and one of the specific antibodies for T lymphocytes, including MAbs to CD3, CD4, and CD8, respectively. (d through f) Dot plots derived from the same cultures stained with anti-EAV N MAb AF488 and antibodies specific for CD3⁺, CD4⁺, and CD8⁺ T lymphocytes, respectively. (g through i) Lymphocytes infected with the MLV strain were stained with anti-EAV nsp1 MAb and antibodies specific for T lymphocytes, including MAbs to CD3, CD4, and CD8, respectively. (j through l) Dot plots derived from the same cultures stained with anti-EAV N MAb AF488 and antibodies specific for CD3⁺, CD4⁺, and CD8⁺ T lymphocytes, respectively.

FIG. 5.

Replication of the VB and MLV strains of EAV in blood-derived monocytes of group A horses. (A) The CD14⁺ monocytes infected with the VB and MLV strains of EAV were examined by dual-color immunofluorescence flow cytometric analysis using MAbs against the EAV nsp1 (12A4) and N (3E2) proteins and MAbs for cell-specific cell surface antigens at 24 hpi. (a, b) Monocytes infected with the VB strain were stained with anti-EAV nsp1 MAb (panel a) or anti-EAV N MAb (panel b) and a MAb specific to cell surface antigen CD14. (c, d) Monocytes infected with the MLV strain were stained with anti-EAV nsp1 MAb (panel c) or anti-EAV N MAb (panel d) and a MAb specific to cell surface antigen CD14. (B) Replication of the VB (black circles) and MLV (white circles) strains in purified monocytes. Tissue culture fluids were collected at the indicated times, and viral titers were determined as the log₁₀ TCID₅₀/50 μl. The values shown are the mean viral titers ± the standard error of the mean. (C) Quantification of viral RNA copy number in cell culture fluid from purified monocytes infected with the VB (black bars) and MLV (white bars) strains using qrRT-PCR. Results are expressed as mean values ± the standard error of the mean.

FIG. 6.

Infection of lymphocytes and monocytes with recombinant EAV strains. The genome of the infectious full-length cDNA clone of rVBS (red boxes) and the genome of the rMLV clone (blue boxes) are depicted. The genes encoding structural proteins of EAV HK116 are shown in green. The four chimeric viruses containing nonstructural and structural protein genes of either rVBS or rMLV are also depicted. L, leader; An, poly(A). CD3⁺, CD4⁺, and CD8⁺ T lymphocytes and CD14⁺ monocytes infected with recombinant viruses rVBS (a through d), rVBS/HK116 S (e through h), rMLV (i through l), rVBS/MLV S (m through p), rMLV/VBS S (q through t), rMLV/VBS 234 (u through x), and rMLV/VBS 56 (y through z″) were examined by dual-color immunofluorescence flow cytometric analysis using MAbs against EAV nsp1 (12A4) and MAbs for cell-specific cell surface antigens at 24 hpi.

FIG. 7.

Predicted membrane topology of minor (GP2, GP3, and GP4) and major (GP5 and M) EAV envelope proteins. Amino acid (a.a.) substitutions that occurred during extensive cell culture passage of the VB strain of EAV, resulting in the HK116 and MLV strains of the virus, are indicated. A predicted model of the disulfide-bonded structure of covalently linked minor envelope proteins GP2, GP3, and GP4 based on previous experimental studies (19, 25, 63-65) is depicted. Intramolecular (solid line) and intermolecular (dotted line) cysteine bridges are depicted. Major envelope proteins GP5 and M are covalently linked by a disulfide bond (S-S) formed between Cys-8 in the M protein and Cys-34 in the GP5 protein (54).