Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

Abstract

Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry. IMPORTANCEThe alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.A variety of enveloped viruses enter host cells by using a core fusion machinery to facilitate the initial receptor-binding step, followed by fusion of the host cell membrane with the virion envelope. Most enveloped viruses use one or two surface glycoproteins to bind to receptor and mediate fusion. In contrast, the herpesvirus core fusion machinery consists of the heterodimeric complex gH/gL and the fusogen gB, with an additional receptorbinding protein such as gD in the alphaherpesvirus pseudorabies virus (PrV) or gp42 in the gammaherpesvirus Epstein-Barr virus (EBV) (1). For infectious entry, PrV requires gB and gH/gL as well as gD, which binds to specific host cell receptors such as nectin-1 and -2 and HveD (2), whereas EBV requires gB, gH/gL, and gp42 for B cell entry, with gp42 binding the receptor...

Pseudorabies virus infection inhibits autophagy in permissive cells in vitro

“…7c). To determine the effects of autophagy on virus entry, we analyzed the entry presences of shRNA transfected cells at different times of penetration according to methods that were previously described3839. The results in Fig.…”

“…After 2 days, the cells were fixed, stained with crystal violet and the plaques were counted as described above in the plaque formation assay. The amount of virus that penetrated the cells was evaluated after the numbers of PFU were determined383957.…”

Gammaherpesvirus entry and fusion: A tale how two human pathogenic viruses enter their host cells

“…Similar to the B cell entrytriggering complex, it is likely that the epithelial cell entry-mediating complex requires conformational changes within gH/gL that enable interaction with gB (Longnecker et al, 2013). Compatible with this hypothesis, the domain interfaces of the four domains and their flexibility are required for gH/gL-activated gB-driven fusion process (Böhm et al, 2015, Chen et al, 2013, Möhl et al, 2015, Wu et al, 2005. In addition, the disulfide bond of DII tightens the syntaxin-like bundles and thereby induces a local rigidity that is required for epithelial cell fusion.…”

“…The overall structure is formed by four structural domains with divergent domain interfaces, whose flexibility is important for gH/gL functioning as a trigger for fusion (Böhm et al, 2015, Chen et al, 2013, Möhl et al, 2015, Wu et al, 2005. Although the crystal structures of KSHV entry proteins have not been solved, the sequence homology between EBV and KSHV glycoproteins, 42% for gB, 26.8% for gH and 27.1% for gL (Gish andStates, 1993, Papadopoulos andAgarwala, 2007), allowed homology modeling of KSHV gB, gH and gL.…”

Structural and Mechanistic Insights into the Tropism of Epstein-Barr Virus

“…The strikingly similar structure of gH/gL, despite the low amino acid conservation, led to the partition into four structural domains with varied domain interfaces due to divergent inter-domain packing angles ( Backovic et al, 2010 ; Matsuura et al, 2010 ). Mutagenesis studies of EBV and PrV gH found that these domain interfaces and their flexibility are important for gH/gL function during fusion ( Böhm et al, 2015 ; Chen et al, 2013 ; Möhl et al, 2015 ; Wu et al, 2005 ). The intimate interaction of gL with the N-terminus of gH forms domain I (D-I) and only a single helix, known as the D-I/D-II-linker-helix, connects D-I with the rest of gH forming a large inter-domain groove ( Matsuura et al, 2010 ).…”