Herpesviruses are loyal companions. Once infected, they remain in the body for a lifetime and can cause new symptoms of disease in the event of stress or a weakened immune system. A common example in humans is the herpes simplex virus type 1, which causes the known cold sores. Herpesviruses also cause childhood chickenpox or Pfeiffer's glandular fever. The viruses can be found in practically all vertebrates. Eight herpesviruses are specific to humans, some of which can cause serious infections and cancer.
The scientists of the Junior Research Group Herpesviruses at the DPZ are investigating the infection mechanism of the human herpesvirus type 8, also known as Kaposi's sarcoma-associated herpesvirus, or KSHV. In immunocompromised patients, the virus can cause Kaposi's sarcoma, a solid tumor, as well as two malignant diseases in which infected B lymphocytes multiply uncontrollably: primary effusion lymphoma and a variant of multicentric Castleman's disease. KSHV-associated tumors occur primarily in the context of HIV infection and are widespread in sub-Saharan Africa.
For the infection of host cells, KSHV uses a receptor protein, EphA2, which is located in the cell membrane. The membrane protein belongs to the family of ephrin receptor tyrosine kinases, of which 14 different variants are known in humans. The virus binds to the receptor protein with a glycoprotein complex in the virus envelope to enter the cell. "KSHV prefers to use the EphA2 receptor as an entry ticket," says Alexander Hahn, head of the Junior Research Group Herpesviruses at the DPZ. "However, we were also able to detect an affinity of the virus for other members of the Eph family of receptors. We wanted to find out whether these receptors also play a role in the infection of B lymphocytes with KSHV. Until now, this was only known for cells growing on a solid substrate, so-called adherent cells". This is also interesting because the mechanisms for the infection of B cells and adherent cells seem to differ fundamentally. While adherent cells can be infected by free virus, an efficient infection of B cells usually only occurs through direct contact between virus-producing cells and B cells.
In interaction studies with glycoproteins of the virus and proteins from a human B cell line, the scientists were able to identify the ephrin receptor tyrosine kinases EphA7 and EphA5 as receptor candidates for KSHV. In order to demonstrate the function of these receptors for infection, the researchers used CRISPR to generate genetically modified B cell lines that no longer contained either one or the other receptor and used them for infection studies.
"In the cell line without the EphA5 receptor, we were able to detect a 57 percent decrease in the rate of infection with KSHV," said Anna Großkopf, PhD student in the Junior Research Group Herpesviruses and first author of the study, summarizing the results. "In the cell lines without EphA7 receptor, the infection rate was even reduced by up to 84 percent. This shows that EphA7 in particular seems to be important for the infection of this B-cell line".
The scientists also repeated the experiments with the rhesus monkey rhadinovirus, RRV for short, which is closely related to KSHV. Here, too, the researchers were able to detect a strongly reduced infection of the cells without EphA7 receptor by up to 99 percent. An analysis of three human B cell lines from effusion lymphomas also showed that the EphA7 protein was present in large quantities in one of the cell lines and the EphA2 receptor in the other two, which suggests that these proteins probably played a role in the development of these B cell lymphomas.
"The study shows for the first time by gene knockout that KSHV uses other ephrin receptors, such as EphA7, in addition to the preferred EphA2 molecule for cell entry," says Alexander Hahn. "In addition, this receptor family seems to play an important role in the infection of B cells – the cell type from which lymphomas can develop. In fact, we see high expression of EphA2 or EphA7 on different cell lines from primary effusion lymphomas, i.e. cells isolated from diseased patients. A deeper understanding of the infection pathways of the virus is essential to better understand disease development and to develop targets for future therapies or vaccination strategies". In the future, the researchers hope to investigate whether their results can also be transferred to other types of B cells.
The first author of the study, Anna Großkopf, was awarded a travel grant from both the International KSHV Workshop in New York and the International Herpes Virus Workshop in Knoxville to present her results there.
Großkopf AK, Schlagowski S, Hörnich BF, Fricke T, Desrosiers RC, Hahn AS (2019): EphA7 functions as receptor on BJAB cells for cell-to-cell transmission of the Kaposi's sarcoma-associated herpesvirus (KSHV) and for cell-free infection by the related rhesus monkey rhadinovirus (RRV). Journal of Virology, 93: e00064-19, doi: 10.1128/JVI.00064-19