Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

Abstract

Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC 5 S mLANA ) complex and mLANA's E3 ligase activity on host NF-B and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease. A s obligatory intracellular parasites, viruses have evolved mechanisms to modulate ubiquitination, which is an essential regulatory mechanism in eukaryotes, controlling a wide range of cellular pathways. Ubiquitination occurs through a threeenzyme cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase enzyme (1). E3 ligases bind to the E2-ubiquitin intermediate and the substrate, catalyzing the transfer of ubiquitin to the substrate target lysine. Many E3 ligases have been described, such as Cullin 5-RING E3 ligases (CRL5), also known as Elongin BC/Cullin 5/SOCS (EC 5 S) E3 ligases. They are multisubunit complexes containing a scaffold protein (Cullin 5) attached to a RING finger protein (Rbx) (Cullin 5-Rbx module), an adaptor heterodimer (Elongin B/C), and a substrate recognition protein (suppressor of cytokine signaling [SOCS] box protein). The last component bridges the substrate of ubiquitination and the E3 ligase complex by interacting with Elongin B/C and Cullin 5 through a SOCS box motif (2-4). IMPORTANCE The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virusCertain viruses encode proteins with SOCS box motifs to hijack the components of cellular E3 ligases, thus modulating the ubiquitination pathway. Examples are latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) (5, 6) and murid he...

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA

“…It is likely that the presence of multiple TR elements (each of which contains LBS1-2) in MHV68 results in enhanced cooperative binding and/or other oligomerization events that increase binding efficiency which may allow kLANA to efficiently mediate episome maintenance of mTR DNA and virus persistence. For instance, despite mutations that engendered severe mLANA DNA binding deficiency to mLBS1-2, mLANA efficiently mediated episome persistence in the setting of a full complement of mTRs in MHV68 [ 62 ]. Further, the finding of large, recombinant 4TR and 8TR episomes here (Figs 1 and 2 ; S1 and S2 Figs), and as previously observed[ 7 , 63 , 64 ] likely reflects the need for ~40 TR elements, similar to that of KSHV, for optimal functional efficiency.…”

Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68

“…Mutation of this region significantly attenuates latency and reduces reactivation (51)(52)(53). mLANA also carries an E3 ligase domain essential for GC B cell expansion (54). Conservation between mLANA and kLANA facilitated studies examining precise domains of kLANA crucial for in vivo function (55)(56)(57) and revealed that kLANA and mLANA are functionally interchangeable for episome maintenance (58,59).…”

“…Nevertheless, NF-κB signaling is likely downregulated during specific phases of infection, as both mLANA and MHV-68 RTA target NF-κB subunit RelA/p65 for degradation. Mutation of the mLANA E3L domain abrogates MHV68-driven GC B cell infection, implicating mLANA E3L activity in GC B cell proliferative expansion ( 54 ). Toll-like receptor (TLR)-induced NF-κB activation disrupts an established latency program to increase genome-positive cells or reactivation in vivo ( 70 ).…”

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire

“…MHV68 LANA targets NF-κB via E3 ubiquitin ligase activity for degradation. Loss of this function enables mLANA to support latency but blocks expansion in the germinal center compartment [ 181 ]. The viral M2 has route-dependent roles in latency establishment and reactivation, and drives IL-10 production and plasma cell differentiation upon expression in primary B cells [ 144 , 163 , 182 ].…”