and have through this part of life.

If it weren’t for my mother Roxanne, brother Billy, and sister-in-law Olya, I would probably not

be here right now. Their support has pushed me to pursue more in my life, and they always made sure I had

a roof over my head and a fridge full of food.

The Pellett Lab has been a nice place to call home with a great family for the last few years. Ian

McCusker showed me the ropes in my early days and started the weekly Saturday lunch tradition. Amina

Wofford was there to hear my crazy ideas, no matter how early in the morning, more than anyone else, and

she made a significant impact on my writing. Jillian Green brought a new side and perspective to the lab

that has benefitted us all. Former members of the lab, Christina Lim, William Close, and Ashley Anderson,

gave me a platform to expand from during my time here. Lastly, Phil Pellett has provided me with the

List of Figures ................................................................................................................................................ v

CHAPTER 1: INTRODUCTION TO HUMAN HERPESVIRUSES ........................................................... 1

Introduction ............................................................................................................................................... 1

Overview ................................................................................................................................................... 1

Herpesviruses ............................................................................................................................................ 1

Herpesvirus taxonomy ............................................................................................................................... 2

HCMV ....................................................................................................................................................... 3

Virion structure.......................................................................................................................................... 3

Replication Cycle ...................................................................................................................................... 5

The cytoplasmic virion assembly compartment ........................................................................................ 5

Glycolysis ................................................................................................................................................ 10

Apoptosis ................................................................................................................................................. 10

Methods ................................................................................................................................................... 11

CHAPTER 3: VALIDATION OF MARKERS FOR cVAC ANALYSIS .................................................. 12

Introduction ............................................................................................................................................. 12

Results and Discussion ............................................................................................................................ 14

CHAPTER 4: DEVELOPING A 3D CELL MODEL FOR cVAC VISUALIZATION ............................. 15

Introduction and Preliminary Data .......................................................................................................... 15

Materials & Methods ............................................................................................................................... 16

Results and Discussion ............................................................................................................................ 16

CHAPTER 5: CONCLUSIONS .................................................................................................................. 18

Appendix ..................................................................................................................................................... 29

References.................................................................................................................................................... 31

ABSTRACT ................................................................................................................................................ 35

Fig. 1: False Colored Electron Micrograph of HSV-1 Virion. .................................................................... 19

Fig. 2: Herpesvirus replication cycle. .......................................................................................................... 20

Fig. 3: Diagram of HCMV cVAC. .............................................................................................................. 21

Fig. 4: Colocalization of nuclear and cellular markers during HCMV infection. ....................................... 22

Much of chapters 1 and 2 is from chapter 10, Betaherpesvirus assembly and egress: Recent

advances illuminate the path, of Advances in Virus Research, Volume 108 "Virus Assembly and Exit

Pathways" [1]. My writing included: the abstract, introduction, virus-modified cellular metabolic shifts and

mitochondrial regulation, apoptosis, and virion envelope structure. The published work includes human

cytomegalovirus (HCMV), the focus of the thesis work, and the human roseoloviruses (human

herpesviruses 6A, 6B, and -7). The following text under the headings Overview, Herpesviruses,

Herpesvirus taxonomy, and Envelope are from the chapter, with some edits and updates.

The human betaherpesviruses include human cytomegalovirus (HCMV) and the human

roseoloviruses (human herpesviruses 6A, 6B, and -7; HHV-6A, HHV-6B, and HHV-7). They are important

emerged from studies of virus-host interactions that have evolved over millennia.

Viruses are obligate, intracellular parasites that can inhabit all forms of cellular life and replicate

in various cell types in their host. They manipulate and modulate host-cell systems to enable their successful

replication. Herpesviruses do this in the context of life-long infections in which the virus establishes a latent,

or quiescent, state during which the virus genome is maintained, but no infectious virions are produced.

reactivates to lytic replication, during which new infectious virions are produced that can be transmitted to

other cells in that individual and can also be transmitted to another susceptible host.

start of the infection.

Members of the Herpesviridae family infect a broad host range of mammals, birds, and reptiles.

More than 300 identified herpesviruses belong to the order Herpesvirales [2], which also includes viruses

of amphibians and fish in the family Alloherpesvirdae, and mollusk viruses in the family

Malacoherpesviridae. Family Herpesviridae is further divided into three subfamilies, the Alpha-, Beta- and

Gammaherpevirinae. Alphaherpesviruses include e.g., herpes simplex viruses 1 and 2 (HSV-1 and -2),

varicella-zoster (VZV), and pseudorabies virus (PRV). Gammaherpesviruses include Epstein-Barr virus

(EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), which are both associated with cancers.

Betaherpesviruses include cytomegaloviruses (CMV) of humans (HCMV), mice/rats (MCMV), guinea pigs

replication occurs in epithelial and endothelial cells, and latency is established in monocytes and T-cells;

roseoloviruses replicate in T-cells and cells of other lineages, and like HCMV, establish latency in lymphoid

cells and monocytes. An uncommon characteristic of HHV-6A and -6B is their ability to integrate into

human chromosomes, occasionally in germ cells, which results in the presence of the virus in every cell of

the offspring. Gammaherpesviruses are generally lymphotropic. EBV and KSHV lytic replication occur in

cause of non-genetic birth defects, child deaths, is associated with various pregnancy complications,

developmental abnormalities, and cognitive disabilities [3-5]. Children infected with HCMV during the

first trimester of pregnancy are at high risk for severe disease. Disease outcomes are less severe as

pregnancy progresses, but fetal infections also become more likely. Many of the molecular and cellular

mechanisms of congenital HCMV are not well understood. HCMV pathology is likely due to drastic

changes in the cellular microenvironment, including, but not limited to, changes in cell signaling,

differentiation capacity, immune response, and metabolism at the maternal/fetal interface [6-10].

As with all herpesviruses, the HCMV virion comprises a dsDNA genome, nucleocapsid,

proteinaceous tegument layer, and a lipid-envelope (Fig. 1).

Writing this chapter began with an outline made by the Pellett lab. Each lab member was assigned

and references. This process involved a significant amount of curiosity and speculation that led to many

interesting possible connections.

Editing was done continuously and was a crucial component of completing this large chapter. Most

of my contributed sections were not co-authored, so the majority of editing was done between myself and

my mentor. The process involved reading through the written material together, usually aloud, and

modifying the text to be clear, engaging, and succinct while following a logical flow.

The AI text editing app Grammarly was invaluable during the writing process. The app is

configurable based on your writing context and your preference in tone. This app acts as a helpful guide to

edit while writing and act as an extra pair of eyes on the material. Grammarly not only helps during the

writing process, but it also helped me become a better writer while unassisted.

During Dr. Ashley Anderson’s dissertation work in the Pellett lab, she was trying to develop a

robust method for quantifying HCMV infected cells with cVACs and establish a temporal window for

biogenesis. This process included validation of the simultaneous use of antibodies against the juxtanuclear

Golgi marker GM130 and the viral nuclear marker IE2 (Immediate-Early Protein 2) so another marker

could be used against her protein of interest HCMV pUL103. The proximity of these markers posed an

issue of fluorescent signal bleed from cytoplasmic GM130 or nuclear IE2 that may interfere with qualitative

and quantitative analysis of the Golgi in infected cells.

To address potential colocalization of our markers, I performed immunofluorescent microscopy

with either IE2 or GM130 in HCMV-infected and mock-infected cells.

100% cytopathic effect (CPE) was reached, cells and supernatant were removed from flasks and centrifuged

at 1280xg for 15 minutes at 4°C. A milliliter of 10% 3x autoclaved milk was used to resuspend the pellet.

The cell suspension was sonicated in a Branson analog 450 sonifier cup horn filled with ice water at an

output control of 10 and 30% duty cycle for 10 seconds, followed by 10 seconds of rest, three times total.

temperature before seeding human foreskin fibroblasts (HFFs) to 80% confluence. The following day, cells

At the end of the infections, the cells were fixed with 300 µl of 4% paraformaldehyde in 1x PBS

for 15 min., autofluorescence quenched with 300 µl of 50 mM ammonium chloride for 15 min.,

permeabilized with 300 µl of blocking buffer (5%, glycine, 10% normal goat serum, 0.1% sodium azide in

1x PBS) containing 0.2% Triton X for 15 min. Samples were blocked with blocking buffer for 1 hour before

addition of 75 µl of primary antibodies (GM130, BD Biosciences, 610823; IE2, Millipore, MAB810)

diluted in blocking buffer for 1 hour. Wells were washed twice for 7 min. in 1x PBS and then 75 µl of

fluorescence-tagged secondary antibody Alexa Fluor 568-conjugated goat anti-mouse IgG,( A-11031,

Invitrogen, Waltham, MA) diluted in blocking buffer for 1 hour was added and then washed twice for 7

Tables, and a linear contrast adjustment was made using a consistent macro for all experimental conditions

in Fig. 4.

The purpose of this work was to assess localization of common cellular and viral markers for

present in the cytoplasm and does not colocalize with nuclear DAPI (Fig. 4). These data indicate that these

nuclear and perinuclear markers are unrelated in localization and can be detected using the same secondary

fluorophore without interfering with the analysis of one or the other.

Any Golgi signal in the nuclear area is likely due to overlap in X and Y planes, but not in the same

Z plane. 3D-microscopy shows this overlap is due to the Golgi-apparatus wrapping above or below the

nucleus (Fig. 5) and not the invasion of immunofluorescent markers. Human interpretation of these images

may seem obvious, but the unbiased computation of these data has added layers of complexity.

Discrete localization of these markers allows the same secondary antibody to detect two separate

signals, allowing for a third marker using a different secondary antibody. Although not shown, it is possible

to segment the IE2-positive nuclei from the IE2 and GM130 positive channel using ImageJ, allowing for

During this process, tissue staining protocols were optimized using commercially acquired HCMV-

positive lung tissue slides (Azer Scientific). Accurate intracellular imaging of these slides proved to be

impossible due to the background fluorescence found in unstained and stained samples (Fig. 7). The

hospital-acquired tissues from other organs of HCMV-positive children are available in small amounts and

are not appropriate for troubleshooting or large-scale replication. Specimen availability quantity and

technical limitations are a major roadblock to this work.

To overcome the issues above, we evaluated use of a three-dimensional cell-based model in place

of sectioned tissue samples using cell spheroids.

Cell spheroids are self-assembling aggregates of cells formed by promoting cell-to-cell interactions

in three dimensions rather than cell-surface interactions in a culture vessel [65] (Fig. 8). When the cells

plates and placed on a rotating shaker to form a concave surface as the agarose cools. Once the agarose is

solidified, cells are be seeded into the wells where they attach to each other rather than the coating on the

bottom of the plate. Cells maintain their typical morphology, and cell-to-cell interactions have an added z-

dimension, similar to the multiple layers of cells in tissues.

from Ibidi Biosciences (https://ibidi.com/img/cms/support/AN/AN32_Generation_of_spheroids.pdf). 1%

Melted agarose in PBS was added to a pre-heated (110F for one hour) multi-well culture plate (Corning

Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate; 09-761-145, Corning, NY). After adding

agarose, the multi-well plate was placed on a rotating shaker to form a concave surface as the agarose

cooled. After complete solidification of the agarose, 15 minutes, cells can be seeded into wells. Cells are

24-Well, Cell Culture-Treated, Flat-Bottom Microplate; 09-761-146, Corning, NY). The seeded cell

density is equal between 3D and 2D cell cultures at 70% confluency, about 70,000 cells/well. 24 hours

dimensional cell-based model.

To verify that this model can support viral infection comparable to normal cell culture, I seeded an

equal number of cells into uncoated or agarose coated wells in a 24-well plate. After 24 hours, the cells

were infected with GFP-tagged HCMV and were imaged over a five-day time course (Fig. 9). GFP-tagged

HCMV infected cells similarly, whether they were in a spheroid or a monolayer when observed under low-

develop the current best workflow and protocols (Fig. 10 and Appendix). The spheroid model is inexpensive

and reproducible. The model has potential to be handled and processed like a tissue specimen or it may also

be imaged using 3D-confocal microscopy to address cVAC biogenesis in a tissue-like structure.

In my current workflow, the spheroids are processed like human tissue, but using 3D-confocal

microscopy, it is also possible to optically section a sample and image the entirety of a spheroid. Confocal

microscopes can image through an entire sample up to 300 microns thick and the spheroids I generate are

typically 100-150 microns in diameter. This method could eliminate the complications of histological

processing and staining, although this method should be technically possible, I haven’t done it, nor have I

seen anyone else do it. If I were to do this process again, I would pursue optical sectioning with microscopy

rather than physical sectioning.

HCMV is a highly prevalent pathogen that causes severe disease in immunocompromised and

egress. This work validated the concurrent use of the same secondary antibody to detect the nuclear viral

marker IE2 with the cellular Golgi-marker GM130 in fluorescence microscopy. The cVAC has not been

well established in human tissues, so we have developed a 3D cell culture model to study HCMV infection

and cVAC biogenesis in a tissue-like environment. I showed that HCMV infection progresses similarly in

cells in a monolayer or spheroid. I have also established methods to generate, infect, and process these

spheroids for immunofluorescent microscopy of cVAC markers.

F for 20-60

Add enough so you can see the spheroid with the naked eye. (50,000 cells/spheroid is a

1. Wash Slides for 3 minutes each in:

2. 100% Alcohol twice

3. Cook at 30% power until the solution boils (only a few seconds).

4. Cook at 20% power for 5 minutes.

2. Circle tissues with hydrophobic barrier pen.

4. Drain off H202, wash in 100-300uL of PBS for 5 minutes.

6. Drain off Blocking Buffer with Triton-X, add 300 uL of Blocking buffer and incubate for 1 hour

11. Prepare Vector Labs TrueVIEW Autofluorescence Quenching Kit.

should be 1:1:1)

13. Drain off TrueVIEW, wash in PBS for 5 minutes.

14. Add mounting medium with DAPI to coverslip, lay slides on their coverslips and allow the medium to

15. Seal the coverslip with clear nail-polish, allow to dry before imaging.

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