The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Abstract

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

Global aspects of viral glycosylation

“…Moreover, the stalk mutation had similar effects in a closely related Nipah virus (Bradel-Tretheway et al 2015). In Hepatitis C Virus, disruption of specific N-glycosites in the two highly N-glycosylated envelope glycoproteins E1 and E2 results in formation of unproductive E1-E2 heterodimers (Meunier et al 1999;Goffard et al 2005;Orlova et al 2015), whereas specific O-glycosites on Hendra virus protein G have been shown to affect association with protein F (Stone et al 2016), suggesting both types of glycans can carry out similar functions. Similarly, Oglycosites have been found in regions of varicella zoster virus gE, important for interaction with partner gI ), but the glycan specific functions are yet to be uncovered.…”

“…In addition to general functions, specific types of glycans on viral glycoproteins often affect receptor binding and cell fusion (Lin et al 2003;Lozach et al 2003;Gramberg et al 2005;Davis et al 2006;Miller et al 2008;Chen et al 2014;Phoenix et al 2016;Wang et al 2016). Moreover, it is becoming increasingly clear that distinct glycans play separate roles in various stages of the viral cycle (Goffard et al 2005;Falkowska et al 2007;Helle et al 2010;Wang et al 2013Wang et al , 2017Lennemann et al 2014;Bradel-Tretheway et al 2015;Luo et al 2015;Orlova et al 2015;Wu et al 2017). Some site-specific functions of viral glycosylation have been assigned for both N-and O-linked glycans mostly by site-directed mutagenesis studies.…”

“…Prominent examples include the Ebola virus glycoprotein modified by a very high content of O-linked glycans, and the HIV-1 glycoprotein gp160 that is glycosylated by the addition of multiple N-linked glycans (Jeffers et al 2002;Pabst et al 2012;Yang W et al 2014). Although specific functional roles have been assigned to distinct glycosites of multiple viruses (Goffard et al 2005;Falkowska et al 2007;Helle et al 2010;Wang et al 2013Wang et al , 2017Lennemann et al 2014;Bradel-Tretheway et al 2015;Luo et al 2015;Orlova et al 2015;Suenaga et al 2015;Stone et al 2016;Wu et al 2017), it is generally believed that the high levels of glycosylation serve as a protective shield from the host's immune system (Francica et al 2010;Helle et al 2011;Sommerstein et al 2015;Behrens et al 2016;Gram et al 2016;Walls et al 2016). The other major role of glycosylation occurs during virus entry where glycans on the host cell represent viral receptors interacting with carbohydrate binding proteins on the viral surface.…”

Viral glycoproteomes: technologies for characterization and outlook for vaccine design

“…Furthermore, the competitive activity of such enzymes in space and time results in the creation of multiple proteoforms carrying an array of different structures and having variable numbers of occupied glycosites . This array of different viral glycan structures not only shields them from host immunity, but is often required for proper viral glycoprotein function . In addition, such a plethora of protein modifications, combined with mutagenesis due to selection pressure, may complicate the creation of potent and lasting vaccines.…”

“…Analysis of O‐linked glycans on HCV E2 revealed considerable microheterogeneity at the six identified glycosites . Mutational analysis of specific N‐glycosites in the two envelope glycoproteins E1 and E2 has identified glycosites important for protein–protein interaction . Moreover, distinct putative N‐ and O‐glycosylation site mutations differentially affected viral particle production, infectivity, CD81 binding, and neutralizing antibody activity .…”