Dissertation
Tropism of human pegivirus (formerly known as GB virus C) and host immunomodulation: insights into viral persistence
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2015
DOI: 10.17077/etd.i61vcmps
Abstract
<p>Human Pegivirus (HPgV; originally called GB virus C) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are infected with HPgV. No causal association between HPgV and disease has been identified; however, several studies found an association between persistent HPgV infection and prolonged survival of HIV-infected individuals that appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (>10 million genome copies/ml plasma) but grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. The overall goals of my thesis were to characterize HPgV tropism, effects of HPgV infection on host immune response and mechanisms of viral persistence.</p>
<p>Previous studies found HPgV RNA in T and B lymphocytes and ex vivo infected lymphocytes produce viral particles. To further characterize HPgV tropism, we quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naïve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) obtained from persistently infected humans using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 9), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naïve (CD45RA+) CD4+ cells than in central memory and effector memory cells (p<0.01). HPgV sequences were highly conserved between patients (0.117 ± 0.02 substitutions per site) and within subjects (0.006 ± 0.003 substitutions per site). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-positive microvesicles (SEV) from serum delivered CFSE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells (PBMCs) with evidence of subsequent viral replication. Thus, HPgV RNA-positive SEV may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.</p>
<p>Although HPgV infection reduces NK cell activation in HIV-infected individuals, the mechanism by which this occurs is not characterized. We studied HPgV effects on NK cell non-cytolytic function in HIV-infected people by measuring expression of IL-12 induced interferon gamma (IFNg) and cytolytic function by measuring K562 target-cell induced CD107a and granzyme B. IFNg expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects treated with combination antiretroviral therapy (p=0.02). In contrast, cytolytic NK cell functions were not affected by HPgV. Inhibition of IFNg was due to inhibition of tyrosine kinase (Tyk2) by HPgV envelope protein E2. HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited IL-12-mediated IFNg release by NK cells. Thus HPgV-E2 inhibits NK cell non-cytolytic functions. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV's ability to persist with high viral loads (>10 million genome copies/ml plasma) and reduce immune cell activation. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.</p>
Details
- Title: Subtitle
- Tropism of human pegivirus (formerly known as GB virus C) and host immunomodulation: insights into viral persistence
- Creators
- Ernest Tafara Chivero - University of Iowa
- Contributors
- Jack T. Stapleton (Advisor)Al Klingelhutz (Committee Member)Chioma Okeoma (Committee Member)Jon Houtman (Committee Member)Nicholas Zavazava (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Spring 2015
- DOI
- 10.17077/etd.i61vcmps
- Publisher
- University of Iowa
- Number of pages
- xvi, 108 pages
- Copyright
- Copyright 2015 Ernest Tafara Chivero
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 93-108).
- Public Abstract (ETD)
Human Pegivirus (HPgV; originally called GB virus C) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are actively infected with HPgV. No causal association between HPgV and disease has been identified; however, several studies found a beneficial association between persistent HPgV infection and prolonged survival in HIV-infected individuals that appears to be related to a reduction in host immune activation. However, the primary permissive cell type(s) infected by HPgV has not yet been identified and mechanisms of viral persistence and host immune modulation remain poorly characterized thus hindering potential applications in treating persistent viral infections and immune activation.
Previous studies found HPgV RNA in T and B lymphocytes. My results show that in addition to these cells, Natural Killer (NK) cells and monocytes from infected humans also have HPgV RNA, implying a broad tropism. I found that serum microvesicles containing HPgV RNA transmit viral RNA to various immune cells resulting in productive infection and contributing to viral spread.
To understand HPgV persistence mechanisms, I examined effects of HPgV on NK cells that play important roles in clearing viral infections. NK cells from HPgV infected people had reduced interferon-ɣ release following IL12 stimulation compared to HPgV uninfected. Inhibition of IFN-ɣ was due to inhibition of tyrosine kinase (Tyk2) by HPgV envelope protein E2. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV persistence and reduced immune activation during HIV-coinfection. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9983776616802771
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