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The Pennsylvania State University The Graduate School HUMAN CYTOMEGALOVIRUS UTILIZES ESCRT-III AND EXTRACELLULAR VESICLES TO ENHANCE VIRUS SPREAD A Dissertation in Biomedical Sciences by Nicholas T. Streck © 2020 Nicholas T. Streck Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2020 The dissertation for Nicholas Streck was reviewed and approved by the following: Nicholas J. Buchkovich Assistant Professor of Microbiology and Immunology Penn State Cancer Institute Dissertation Advisor Chair of Committee John W. Wills Distinguished Professor Emeritus of Microbiology and Immunology Jeffery T. Sample Professor of Microbiology and Immunology Penn State Cancer Institute Neil D. Christensen Professor of Pathology and Laboratory Medicine Microbiology and Immunology Penn State Cancer Institute Hui-ling Chiang Professor of Cellular and Molecular Physiology Ralph L. Keil Associate Professor of Biochemistry and Molecular Biology Chair of Biomedical Sciences Graduate Program ii Abstract Human cytomegalovirus (HCMV) is a highly prevalent human pathogen that can cause disease in the developing fetus and immunocompromised patients. A better understanding of the replication cycle of the virus may lead to better therapeutic targets to help control infection. During HCMV infection, cellular membrane from vesicles and organelles localize to a perinuclear region within the cell, which is termed the cytoplasmic viral assembly compartment (cVAC). This is the site of envelopment of progeny virions. Both viral and cellular proteins have been suggested to be involved during this process, but the mechanism of HCMV envelopment within the cVAC remains unknown. Many enveloped viruses utilize the endosomal sorting complexes required for transport (ESCRT) proteins for viral budding and envelopment. The ESCRTs are required for the membrane scission event during intraluminal vesicle (ILV) formation. The late ESCRTs, ESCRT-III and vacuolar sorting-associated protein 4 (VPS4), are responsible for membrane constriction and vesicle formation. Previous data suggest a potential role for the late ESCRTs during HCMV infection, but it is unclear whether the ESCRTs promote HCMV envelopment or a different stage of the replication cycle. We hypothesized that ESCRT-III and VPS4 are required for HCMV envelopment at the cVAC. The role of the late ESCRTs was investigated through the inducible expression of dominant negative ESCRT subunits to inhibit ESCRT activity at various stages of HCMV infection. We found that ESCRT-III was not required for final envelopment of HCMV, as enveloped intracellular and extracellular virions could be found in infected cells when ESCRT activity was blocked. However, expression of iii dominant negative ESCRT-III subunits slowed HCMV spread during a low multiplicity of infection (MOI). Cells release signaling molecules inside membrane-enclosed compartments termed extracellular vesicles (EVs) that transfer proteins and nucleic acids to recipient cells. Virus infection commonly alters this signaling pathway by affecting the signaling molecules loaded as cargo into EVs. Changes in EV composition will impact whether they inhibit virus infection or promote an environment more conducive to replication and spread. EV biogenesis and release can occur through a mechanism that involves the ESCRT machinery. We hypothesized that inhibition of ESCRT activity may slow HCMV spread by preventing the release of proviral EVs that enhance virus spread to uninfected cells. We found that HCMV upregulates ESCRT protein expression during infection, which coincides with increased EV biogenesis. Purification of EVs from HCMV-infected fibroblasts identified that infection increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs purified from infected cells also contained viral factors known to be important for the initiation of the early stages of infection. The importance of EVs on HCMV spread was confirmed as treatment with the EV inhibitor GW4869 also slowed virus spread, and the transfer of EVs purified from HCMV-infected cells increased the efficiency of HCMV spread. Together, this work shows that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection. iv Table of Contents List of Figures............................................................................................................... viii List of Tables................................................................................................................ x List of Abbreviations................................................................................................... xi Preface........................................................................................................................... xv Acknowledgments........................................................................................................ xvi Chapter 1: Introduction.............................................................................................. 1 Human cytomegalovirus.................................................................................... 2 HCMV clinical significance.................................................................. 4 HCMV entry.......................................................................................... 6 HCMV gene expression and genome replication.................................. 8 HCMV assembly and envelopment....................................................... 10 HCMV egress and spread...................................................................... 14 The ESCRT machinery...................................................................................... 16 Membrane scission................................................................................. 16 ESCRT complexes and their function.................................................... 19 ESCRTs and their role during viral infection........................................ 26 Viral late domains.................................................................................. 30 Herpesviruses and the ESCRT machinery............................................. 33 ESCRT-independent viral envelopment................................................ 34 Coronavirus envelopment...................................................................... 35 Extracellular vesicles......................................................................................... 36 Identification of EVs and their biological relevance............................. 36 EV biogenesis........................................................................................ 39 EV composition and cargo selectivity................................................... 41 EVs and virus infection......................................................................... 43 Hypothesis......................................................................................................... 45 v Chapter 2: Nonenvelopment Role for the ESCRT-III Complex during HCMV Infection......................................................................................................................... 46 Abstract............................................................................................................... 47 Importance.......................................................................................................... 48 Introduction........................................................................................................ 49 Results................................................................................................................ 52 Chapter 3: Human Cytomegalovirus Utilizes Extracellular Vesicles to Enhance Virus Spread................................................................................................................. 69 Abstract............................................................................................................... 70 Importance.......................................................................................................... 71 Introduction........................................................................................................ 72 Results................................................................................................................ 74 Chapter 4: Discussion.................................................................................................. 91 Differential regulation of ESCRT proteins during HCMV infection................. 95 Localization of ESCRT-III and VPS4 during HCMV infection 95 ESCRT-III upregulation during HCMV infection 97 Cytotoxic effects of blocking ESCRT activity................................................... 98 HCMV envelopment.......................................................................................... 100 Viral proteins as functional homologs of ESCRT-III subunits 100 HCMV envelopment through other cellular proteins 101 HCMV modulates EV morphology and composition........................................ 103 HCMV infection alters EV size distribution 103 Selective incorporation of viral factors into EVs 104 The role of HCMV glycoproteins on the surface of EVs 106 Potential effects of EVs on recipient cells 107 ESCRT-dependent and ESCRT-independent EVs enhance HCMV spread...... 108 ESCRT and ceramide-dependent biogenesis 108 CD63-dependent biogenesis 110 The role of EVs in vivo.....................................................................................

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