Abstract
Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.
Highlights
The human γ-herpesviruses Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV) distinguish themselves from the α-herpesviruses (herpes simplex virus 1(HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV)) and β-herpesviruses (human cytomegalovirus (HCMV), human herpesvirus 6A/6B/7 (HHV-6A/6B/7)) by their association with a large number of malignancies [1,2,3,4]
While earlier investigations relied on bulk [45,46] or clonal sequencing [47] to determine the ratio of wild-type to drug-resistant viruses, we developed a next-generation sequencing (NGS) approach to accurately quantify the composition of the viral population in herpesvirus dual infection competition assays [48]
We investigated the mechanism of murine γ-herpesvirus-68 (MHV-68) resistance to nucleoside (GCV), nucleotide (CDV, HPMP-5azaC, HPMPO-DAPy) and pyrophosphate analogues (PFA) and evaluated the impact of mutations associated with drug resistance on viral replication capacity
Summary
The human γ-herpesviruses Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV) distinguish themselves from the α-herpesviruses (herpes simplex virus 1(HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV)) and β-herpesviruses (human cytomegalovirus (HCMV), human herpesvirus 6A/6B/7 (HHV-6A/6B/7)) by their association with a large number of malignancies [1,2,3,4]. The human γ-herpesviruses Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV) distinguish themselves from the α-herpesviruses EBV is associated with the development of several lymphoproliferative disorders (Burkitt’s lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma and lymphoproliferative disease in immunocompromised hosts) and carcinomas of epithelial origin (nasopharyngeal carcinoma and gastric carcinoma) [5]. Though EBV and KSHV oncogenesis is mostly associated with latent gene expression, recent investigations demonstrate a significant contribution of lytic replication to viral tumorigenesis [7,8]. Antiviral therapy intended at inhibiting the expression of lytic viral proteins should be beneficial for controlling the early stages of EBV-associated malignancies. The EBV immediately early (IE) proteins BZLF1 and BRLF1 promote interleukin-6 (IL-6) secretion in lytically infected cells, sustaining early lymphoproliferative disease [11] as IL-6 (a cytokine playing a key role in maintenance of immune functions, stimulation of hematopoietic cells differentiation and persistence of inflammation) is a decisive element in various epithelial and hematological malignancies
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