Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner

Abstract

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8 + T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/β/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp 398 and Leu 414 , respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp 184 and Leu 173 , respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection. PLOS PATHOGENSPLOS Pathogens | https://doi.org/10.1371/journal.ppat.

Monoclonal antibodies targeting nonstructural viral antigens can activate ADCC against human cytomegalovirus

“…We also investigated the ability of the UL141 mAbs to promote the control of virus using a recently developed 10-day viral dissemination assay (VDA), which captures the effects of both cytotoxic and noncytotoxic virus control in a fully autologous system ( Figure 7, D and E , and refs. 54 , 55 ). The UL141 mAbs demonstrated a striking ability to enhance NK-mediated virus control in this assay, confirming that they can act as powerful effectors for long-term control of virus infection, even at low effector/target (E/T) ratios.…”

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay

“…It is interesting to note that anti-HCMV activity of monocyte derived macrophages has been observed and that this was not mediated by IFNγ nor primary IFNα or IFNβ although the identity of the secreted factor responsible for the activity was not determined (Becker et al, 2018 ). Other studies have shown that even in the presence of IFNγ or IFNα or IFNβ neutralizing antibodies, HCMV infection can still be controlled by lymphocytes in a non-cytotoxic manner, with granzymes implicated in control (Shan et al, 2020 ). A non-biased mass spectrometry-based approach is likely to be required to elucidate the key antiviral components of effective secretomes.…”

Dual roles of granzyme B

“…45 Gerada et al identified that GZMB cleaved multiple herpes simplex virus (HSV) type 1 and varicella-zoster virus (VZV) gene products (HSV-infected cell protein (ICP) 4, HSV ICP27, VZV open reading frame (ORF) 62 and VZV ORF4), which demonstrate important roles in viral replication. 46 Furthermore, GZMB induces the cleavage of viral immediate-early (IE) proteins IE1 and IE2 in human cytomegalovirus (HCMV)infected cells, 47 which are vital for triggering a temporally coordinated cascade of transcriptional events that lead to expression of early and late viral proteins essential for HCMV reactivation and production of infectious virus. 48,49 When Jurkat cells infected with the vaccinia virus were subjected to treatment with GZMB, a halt in viral protein synthesis and a decrease in the production of infectious new virions occurred because GZMB induced the cleavage of eukaryotic initiation factor 4 gamma 3.…”

“…Additionally, GZMB that enters the cells mainly plays a significant role in eliminating tumour cells/virus-infected cells, [27][28][29][37][38][39][40]44 eliminating intracellular bacteria and parasites, 19,20,41,42 and viruses. [46][47][48][49][50] However, extracellular GZMB plays an important role in inducing the occurrence and development of diseases (such as inflammation, 8,84,[93][94][95][96][97][98][99] fibrosis, 88,115 CAV, 83 AAA, 89 autoimmune blistering diseases, 92,93,108 age-related macular degeneration, 16 SLE/LN, 109 RA, 4 SSPE, 110 cardiovascular disease, [112][113][114] and wound healing [117][118][119][120] ).…”