truck Domselaar for technical assistance and Dr. (main) cytotoxic lymphocytes can block HCMV dissemination impartial of host cell death, and interferon-//. 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 Asp398 and Leu414, 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 Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data show that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV contamination, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic contamination. Author summary Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects, can trigger disease in immune-compromised patients, and plays functions in cancer development. Cytotoxic lymphocytes kill HCMV-infected cells via releasing a set of five cytotoxic serine proteases called granzymes. However, the killing capacity of cytotoxic 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 contamination in a noncytotoxic manner. Here, we show that cytotoxic lymphocytes can also use granzymes to inhibit HCMV replication in absence of cell death. All five granzymes cleave and inactivate both viral immediate-early (IE1/2) proteins, which are essential players for initiating HCMV contamination. Our data support the model that cytotoxic cells employ granzymes to dampen HCMV replication prior to accumulation of sufficient hits to kill the infected cell. Introduction Human cytomegalovirus (HCMV) is usually a member of the beta-family with worldwide seroprevalence of up to 90% [1]. It is the most frequent viral cause of congenital defects and HCMV may promote tumor development [1, 2]. Primary contamination induces a life-long latent contamination, in bone marrow-resident precursor cells of the myeloid lineage (CD34+ hematopoietic progenitor cells), amongst others [3]. Differentiation of these latently infected myeloid precursors into migrating macrophages or mature dendritic cells is the proposed mechanism for viral organ dissemination and reactivation from latency [3]. HCMV replication is normally controlled by a vigorous host immune response [1]. However, in the absence of an adequate immune response, killing capacity of cytotoxic T cells is limited in that multiple hits by T cells are needed to kill a single CMV-infected cell ID1 [18]. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV contamination in a noncytotoxic manner. In the present study, we demonstrate that (donor-derived HCMV-specific) CD8+ T cells and NK cells can inhibit HCMV replication in the absence of host cell death and impartial of IFN-//. Prior to killing, cytotoxic lymphocytes induce the degradation of IE proteins in HCMV-infected cells. We also show that all human granzymes can directly target and cleave viral IE1 and/or IE2, likely to inactivate their function and subsequent HCMV replication. Thus, besides inducing apoptosis, cytotoxic lymphocytes can also utilize noncytotoxic ways to control HCMV contamination, which may be explained by granzyme-mediated targeting of indispensable viral proteins during the earliest phase of the HCMV replication cycle. Results Cytotoxic lymphocytes can inhibit HCMV dissemination in a noncytotoxic manner and can induce IE degradation in infected cells Recently, it has been exhibited by intravital imaging that this killing capacity of cytotoxic T cells is limited, in that multiple T cell hits are required to kill.(G, H) Immunofluorescence images of HeLa cells transfected with HA-tagged IE2, IE21-184, and IE2185-579 (G), HA-tagged IE2, IE21-173, and IE2174-579 (H) constructs. are directly proteolyzed by all human granzymes, with GrB and GrM Eprinomectin being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, 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 Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data show that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV contamination, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic contamination. Author summary Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects, can trigger disease in immune-compromised patients, and plays functions in cancer development. Cytotoxic lymphocytes kill HCMV-infected cells via releasing a set of five cytotoxic serine proteases called granzymes. However, the killing capacity of cytotoxic 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 contamination in a noncytotoxic manner. Here, we show that cytotoxic lymphocytes can also use granzymes to inhibit HCMV replication in absence of cell death. All five granzymes cleave and inactivate both viral immediate-early (IE1/2) proteins, which are essential players for initiating HCMV contamination. Our data support the model that cytotoxic cells employ granzymes to dampen HCMV replication prior to accumulation of sufficient hits to kill the infected cell. Introduction Human cytomegalovirus (HCMV) is usually a member of the beta-family with worldwide seroprevalence of up to 90% [1]. It is the most frequent viral cause of congenital defects and HCMV may promote tumor development [1, 2]. Main contamination induces a life-long latent contamination, in bone marrow-resident precursor cells of the myeloid lineage (CD34+ hematopoietic progenitor cells), amongst others [3]. Differentiation of these latently infected myeloid precursors into migrating macrophages or mature dendritic cells is the proposed mechanism for viral organ dissemination and reactivation from latency [3]. HCMV replication is normally controlled by a vigorous host immune response [1]. However, in the absence of an adequate immune response, killing capacity of cytotoxic T cells is limited in that multiple hits by T cells are needed to kill a single CMV-infected cell [18]. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV contamination in a noncytotoxic manner. In the present study, we demonstrate that (donor-derived HCMV-specific) CD8+ T cells and NK cells can inhibit HCMV replication in the absence of host cell death and impartial of IFN-//. Prior to killing, cytotoxic lymphocytes induce the degradation of IE proteins in HCMV-infected cells. We also Eprinomectin show that all human granzymes can directly target and cleave viral IE1 and/or IE2, likely to inactivate their function and subsequent HCMV replication. Thus, besides inducing apoptosis, cytotoxic lymphocytes can also utilize noncytotoxic ways to control HCMV contamination, which may be explained by granzyme-mediated targeting of indispensable viral proteins during the earliest phase of the HCMV replication cycle. Results Cytotoxic lymphocytes can inhibit HCMV dissemination in a noncytotoxic manner and can induce IE degradation in infected cells Recently, it has been exhibited by intravital imaging that this killing capacity of cytotoxic T cells is limited, in that multiple T cell hits are required Eprinomectin to kill a single CMV-infected cell [18]. This raises the question whether cytotoxic cells can also control HCMV contamination.