Epstein-Barr virus (EBV) may sound unfamiliar, but chances are you have already heard a lot about it. This virus is the primary cause of infectious mononucleosis, more commonly known as mono. More than 90% of adults have antibodies against it, indicating a past or current infection (CDC, 2014). EBV is a member of the Herpesviridae family and has a double-stranded DNA genome that encodes for approximately 100 genes. The virus predominantly invades epithelial cells and B-cells of the human body. Similar to other herpes viruses, EBV can integrate its DNA into the host genome, leading to lifelong latent infections (Odumade et al., 2011). One way the EBV has gotten so good at infecting humans is through its ability to evade the immune system.
Previous research has shown that the EBV genome encodes for proteins that interfere with cellular proteins responsible for antigen presentation to T-cells. These cellular proteins are called major histocompatibility complexes (MHC). There are two classes, MHC class I and MHC class II, and each class serves a different purpose. MHC class I is responsible for presenting antigens to CD8+ T-cells that directly kill pathogens or virally infected cells, while MHC class II presents antigens to CD4+ T-cells that activate B-cells to start antibody production (Murphy, 2012). The EBV proteins that interfere with MHC were predominantly produced in the immediate early or early stage of the lytic cycle. However, the virally infected cells were still able to evade T-cell identification during the late lytic stage. So how were the infected cells able to accomplish this?
Figure 1. Surface levels of the HLA types detected by flow cytometry when coexpressed with BDLF3 (dotted line) or expressed with the control (solid line). Gray shaded area represents background staining with an isotype control antibody. Quinn et al., 2016
In a study by Quinn and colleagues, they first tackled this question by using a plasmid to express 25 different EBV late lytic genes in a cancerous epithelial cell line. They found that the BDLF3 gene, whose function was previous unknown, was affecting the surface expression of both MHC class I and II. Researchers subsequently tested a known line of EBV-positive cells to corroborate that the protein was produced in the late lytic stage. Further tests confirmed that the MHC class I and II were significantly reduced in a epithelial cell line and a B-cell line while the surface levels of other cellular proteins like the transferrin receptor, ICAM and CD19 were not significantly affected.
Since humans have different MHC class I and II alleles, researchers questioned if certain alleles are more affected by BDLF3 than others. The results showed all alleles are affected equally as seen in Figure 1. The decrease in MHC class I (HLA-A2, HLA-B35, and HLA-Cw1) caused by BDLF3 was similar in all three alleles tested and the same results were seen for both types of MHC class II alleles tested (HLA-DR and HLA-DQ).
Knowing that BDLF3 reduces both classes of MHC in virally infected cells, researchers confirmed that the abnormally low surface MHC caused decreased T-cell recognition by measuring the levels of IFN-γ produced by activated T-cells. Cells that expressed both BDLF3 and the antigen the CD8+ or CD4+ T-cells recognize showed significantly lower levels of IFN-γ produced compared to the control group that was expressing the antigen and a control plasmid.
Figure 2. A) Percent of MHC class I or II remaining on the cell surface over time while expressing BDLF3 or a control plasmid. B) Percent of new MHC class I or II arriving at the cell surface while expressing BDLF3 or a control plasmid. Quinn et al., 2016.
Once researchers knew BDLF3 reduced the amount of both MHC classes and that this affected the ability of T-cells to recognize EBV infected cells, the next step was to determine the mechanism behind it. Researchers first determined that BDLF3 selectively affects MHC on the surface of the cell compared to the MHC throughout the whole cell. Next, BDLF3-expressing cells were monitored over time to see if the viral protein is better at internalizing MHC from the surface through endocytosis or if it is more efficient at preventing MHC from reaching the surface in the first place. The results indicate that both are occurring, as seen in Figure 2. The cells expressing BDLF3 had lower surface MHC class I and class II remaining over time and they also had less MHC class I and class II appear on the surface when compared to the control cells.
Figure 3. Surface levels of MHC class I (A) and MHC class II (B) detected by flow cytometry while expressing BDLF3 (dotted line) or a control plasmid (solid line) after incubation with no drug or with the proteasomal inhibitor MG132. Quinn et al., 2016.
Furthermore, cells expressing BDLF3 were either incubated with or without MG132, a proteasomal inhibitor, to establish if BDLF3 uses the proteasome pathway to decrease surface MHC. When incubated without MG132, surface amount of MHC class I and II were reduced as expected, however when incubated with MG132, there was no significant decrease (Figure 3). Researchers lastly found that the MHC class I and II are tagged with ubiquitin, which is necessary for the proteasome degradation. So, results demonstrate that the proteasome pathway is used by BDLF3 to degrade both MHC class I and II from the surface.
Prior to this study, the function of the EBV protein BDLF3 was unknown. Now, BDLF3 is understood to reduce the amount of host cell’s surface MHC class I and II by using the proteasome pathway. BDLF3 allows the infected cells to persist in the host because it evades recognition by both CD8+ and CD4+ T-cells. These new findings are significant because BDLF3 could be a potential drug target to treat EBV. If BDLF3 is targeted and deactivated by a drug, the CD8+ and CD4+ T-cells can then recognize the infected cells and decrease the viral load of the host. This treatment would not eliminate the virus completely, but it could help to decrease the symptoms of EBV. Overall, the understanding of BDLF3 function is exciting because it can lead to further knowledge of strategies the EBV uses to evade immune recognition.
Click here to check out the primary article about BDLF3
References:
“Epstein-Barr Virus And Infectious Mononucleosis”. Cdc.gov. N.p., 2016.
Murphy, Kenneth et al. Janeway’s Immunobiology. New York: Garland Science, 2012.
Odumade, O. A., K. A. Hogquist, and H. H. Balfour. “Progress And Problems In Understanding And Managing Primary Epstein-Barr Virus Infections”. Clinical Microbiology Reviews 24.1 (2011): 193-209.
Quinn, Laura L. et al. “The Missing Link In Epstein-Barr Virus Immune Evasion: The BDLF3 Gene Induces Ubiquitination And Downregulation Of Major Histocompatibility Complex Class I (MHC-I) And MHC-II”. J. Virol. 90.1 (2015): 356-367.
Stonehill Virology 2016 