Feng Lab

HEV infections are usually self-limited, but the infections frequently persist when the immune system is compromised and if left untreated, can lead to serious liver disease. HEV exists in two distinct virion forms: naked virions (nHEV) that are shed into feces and mediate virus transmission between hosts, and quasi-enveloped HEV (eHEV) virions that circulate in the bloodstream and mediate virus spread between cells. The eHEV particles lack viral antigens on their surface, thus they are resistant to circulating HEV-specific antibodies. We previously show that eHEV particles enter cells via a novel entry mechanism that involves lysosomal degradation of the viral envelope. Our recent data show that HEV-specific IgG, but not IgM, effectively block eHEV-mediated spread in cell culture. Our central hypothesis is that antibodies neutralize eHEV intracellularly by preventing virus uncoating in the endosome/lysosome where the viral membrane degrades. Antibodies generated by natural HEV infection and vaccination with truncated HEV capsid proteins (CP) are highly protective against HEV infection, while anti-HEV antibody titers are usually low in patients with chronic HEV infection. Thus, antibodies may have the potential to prevent or treat chronic HEV infection. Despite these encouragements, there are several significant roadblocks. First, the C terminus of the HEV CP, which is not present in the current vaccine and the fecal virus, is intact in the eHEV particles. This is important since structural modeling suggests that the presence of the C terminus of CP significantly alters the surface structure of the virion which likely makes vaccine-induced antibodies less effective against eHEV. Second, our recent work indicates that HEV produces a capsid decoy that is secreted from infected cells in a large quantity and interferes with antibody-mediated neutralization. Third, antibody uptake by hepatocytes is an inefficient process. Here we propose to overcome these obstacles. Aim 1 will test the hypothesis that antibodies targeting virions with intact CP will block eHEV-mediated spread more efficiently. We will determine the structure of authentic HEV virions with intact or cleaved CP and assess if antibodies targeting virions with intact CP neutralize eHEV more efficiently. We will also determine if glycoengineered antibodies with enhanced lysosomal targeting neutralize eHEV more efficiently. Aim 2 will test the hypothesis that neutralizing antibodies that do not bind or bind poorly to the decoy will block HEV spread more efficiently. We will also determine the structure of the CP decoy in complex with antibodies by cryo-EM to gain a better understanding of the evasion mechanism by the decoy. The completion of the proposed work will provide novel insights into the role of antibodies in HEV infection and inform strategies to prevent or cure chronic HEV infection. Click here to learn more about this project

Our Goal
The goal is to better understand the role of antibodies in HEV infection and determine if antibodies can prevent or cure chronic HEV infection.

Funding
This work has been funded through National Institutes of Health grant R01AI175800.

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide and can often persist in immunocompromised individuals leading to significant morbidity and mortality. Currently, there are no FDA- approved vaccines or HEV-specific therapy in the U.S. Our long-term goal is to elucidate the molecular details of the HEV infectious cycle to aid in the rational design of HEV-specific prevention and treatment. HEV is an enterically transmitted positive-strand RNA virus with a unique life cycle: the virus is shed as naked particles into the feces but circulates as quasi-enveloped (eHEV) particles in the bloodstream. The dogma in the field has been that eHEV mediates virus spread in the infected host, and its biogenesis requires the viral ORF3 protein. It is proposed that ORF3 usurps the cellular endosomal sorting complex required for transport (ESCRT) machinery to acquire an envelope from the multivesicular bodies (MVBs). However, in cell culture, HEV can spread regardless of ORF3 expression, and clinical isolates bearing ORF3 start codon mutations have also been described. Thus, the release mechanism for HEV is likely to be more complex than previously thought. In HEV- infected polarized human hepatocyte cultures and human liver chimeric mice, ORF3 almost exclusively localizes to the apical/canalicular membrane. Moreover, our recently published work shows that ORF3 is required for apical but not the basolateral release of HEV from polarized human hepatoma cells. This application seeks to better define the mechanism for HEV release from polarized hepatocytes and to clarify the role of ORF3 in this process. Our central hypothesis is that ORF3 dictates apical release of HEV but is not required for basolateral release from polarized hepatocytes. We propose two specific aims to test this hypothesis using in vitro and in vivo models. Aim 1 will use a highly polarized human hepatoma cell line to elucidate the mechanism for HEV release from both the apical and basolateral sides of human hepatocytes. We will determine that ORF3 recruits HEV capsid and ESCRT components to the apical recycling endosomes (AREs)-derived transport vesicles to facilitate HEV envelopment and exit, and an ORF3-independent mechanism is responsible for HEV release at the basolateral surface that mediates virus spread. Aim 2 will investigate the HEV release mechanisms in a newly developed rat model of HEV infection. The role of ORF3, ESCRT, MVBs and AREs will be investigated using various viral mutants. The morphology and protein composition of HEV virions in the bloodstream will be characterized and compared to virions released from the apical and basolateral sides of polarized human hepatoma cells. The completion of this work will shed new light on the unusual HEV life cycle and clarify the role of ORF3 in HEV infection. The knowledge gained from this research will also have broad implications for hepatocyte biology and noncytolytic release mechanisms for both nonenveloped and enveloped viruses.

Our Goal
The goal is to define the mechanism for HEV release from polarized hepatocytes and clarify the role of the viral ORF3 protein in this process.