Zongdi Feng - US grants

? DESCRIPTION (provided by applicant): Hepatitis A virus (HAV) and hepatitis E virus (HEV) infections are common causes of epidemic food-borne and water-borne hepatitis in under-developed regions of the world. Recently sporadic HEV infections have emerged as a significant public health hazard in well-developed countries. The recognition of zoonotic and blood-borne HEV transmission and its ability to persistent in immunosuppressed individuals is also alarming. Approximately 20% of the U.S. population is seropositive for HEV, indicating that HEV exposure is more common than previously thought. Although both recognized as non-enveloped viruses, recent studies show that HAV and HEV circulate in the blood as quasi-enveloped particles. These novel particles differ from classic enveloped viruses in that no viral antigens are present on the surface of their membrane, therefore are highly resistant to neutralizing antibodies. The impact of this quasi-envelopment on hepatitis virus life cycle, immunity, and pathogenesis is poorly understood. This proposal will explore the mechanisms by which quasi- enveloped HAV and HEV enter the cells and are neutralized by antibodies. Aim 1 will test the hypothesis that entry of quasi-enveloped HAV and HEV is through a cellular mechanism for extracellular vesicular uptake. Aim 2 will determine the mechanism of neutralization for quasi-enveloped HAV and HEV. Completion of these aims will address several of the most significant gaps in our understanding of the quasi-enveloped virus life cycle and pathogenesis.

ABSTRACT The enterically transmitted hepatitis E virus (HEV) has a unique dual life style: while shed as non-enveloped virions in feces, it circulates in the bloodstream as quasi-enveloped virions (eHEV). While the quasi- envelopment provides effective protection for free virions against antibodies, our new data suggest that this strategy is not perfect because antibodies are able to neutralize eHEV intracellularly and limit virus spread. In addition, large quantities of viral RNA-free capsid antigen (free antigen) are present both in the supernatant of HEV-infected cells and in the sera of HEV-infected humans and macaques for reasons that are not under- stood. Our preliminary data suggest that the HEV free antigen is produced as a result from translation initiated at an alternative start codon of the capsid gene and is able to reduce antibody-mediated neutralization of HEV. However, it is not essential for the HEV life cycle. We hypothesize that HEV free antigen serves as an antigen decoy to further dampen the host antibody response during HEV infection. Our long-term goal is to better un- derstand the mechanisms by which HEV free Ag modulates host immunity and contributes to HEV pathogene- sis. The objectives of this project are to elucidate the mechanism(s) underlying the bulk accumulation of HEV free antigen (Aim 1) and to determine if free antigen functions as an antigen decoy (Aim 2). The completion of this study will fill a significant gap in our understanding of the life cycle and pathogenesis of HEV.

Summary Hepatitis E virus (HEV), an enterically transmitted virus, exists either as naked virions that are shed in the feces or quasi-enveloped virions that circulate in the blood. The quasi-envelopment of HEV facilitates noncytolytic release of virus from infected cells. However, the lack of viral envelope proteins raises question about mechanism for HEV spread and the role of antibodies in natural infection. HEV infects ~20 million people annually and causes significant morbidity and mortality. Persistent HEV infection in immunocompromised individuals can lead to rapid progression of liver fibrosis. Currently there are no FDA-approved diagnostics and HEV-specific therapies. Our long-term goal is to unravel the functional role(s) of this unusual envelopment in the virus life cycle and pathogenesis for the purpose of identifying novel targets for therapeutic intervention. The overall objective of this project is to understand the role of the quasi-envelopment in HEV spread and how it affects antibody-mediated neutralization. The viral ORF3 protein is known to play a key role in HEV envelopment. Our preliminary data suggest that ORF3 is subjected to regulation by palmitoylation to mediate virion release. In addition, we found evidence that neutralizing antibodies block HEV spread in cells that have already established infection. Our central hypothesis is that ORF3-mediated quasi-envelopment is required for HEV release into the bloodstream and into the bile (then shed into feces), and quasi-enveloped HEV particles mediate spread with the liver via a novel entry mechanism that is susceptible to post-entry neutralization by antibodies. We will test this hypothesis in polarized hepatocyte cell culture and in human liver chimeric mice. Aim 1 will define the role of palmitoylation of ORF3 in its cellular localization and function in virus release. Aim 2 will define the cellular requirements and role of ORF3 in HEV spread. Aim 2 will also define the role and mechanism of antibodies in blocking HEV spread. The expected outcomes of the proposed research are novel insights into the spread mechanism for a quasi- enveloped virus, and may have implications for other non-enveloped viruses such as hepatitis A, poliovirus, and enteroviruses that also become quasi-enveloped at certain stages of their life cycle. This work will fill gaps in our understanding of the quasi-envelopment processes and how it influences virus spread, pathogenesis and immunity.

Abstract The enterically transmitted hepatitis E virus (HEV) infects ~20 million people annually. HEV infection is usually self-resolving but can persist in individuals with a weakened immune system and result in fast progression into liver cirrhosis. HEV encodes a single capsid protein ORF2. Nonetheless, recent studies show that most ORF2 proteins released from HEV-infected cells are not associated with virus particles. The exact role(s) of the secreted ORF2 (ORF2s) plays is poorly understood, which hampers our understanding of HEV infection and pathogenesis. Our recently published work demonstrated that ORF2s and the capsid are two different translation products. A signal sequence unique to ORF2s directs its secretion via the secretory pathway, where as a conserved, but previously unrecognized, internal start codon is responsible for translation of the capsid- associated ORF2 (ORF2c). We further found that ORF2s exists as a glycosylated dimer with substantial antigenic overlap with the virion, and purified ORF2s was able to inhibit antibody-mediated neutralization of HEV. Based on these data, we hypothesize that ORF2s acts as a decoy to evade the host antibody response during HEV infection. Our long-term goal is to better understand how ORF2s modulates HEV infection and host immunity and whether it contributes to pathogenesis. The objectives of this project are to use an established rat model to test the in vivo role(s) of ORF2s in HEV infection. Aim 1 will determine if eliminating ORF2s expression alters host antibody responses and the course of acute HEV infection. Aim 2 will determine if serum ORF2s interferes with antibody therapy of chronic HEV infection, and if so whether the effectiveness of antibody therapy will be improved by using antibodies that target ORF2c but not ORF2s. The concept that ORF2S functions as an immunological decoy is novel and may have important implications for HEV immune evasion and persistence. The expected outcomes will fill a significant gap in our understanding of the role of secreted ORF2 in the HEV life cycle and pathogenesis with the potential for more targeted therapies where no cure currently exists.