Human cytomegalovirus (HCMV), a beta-herpesvirus, is an important opportunistic pathogen that primarily affects individuals with compromised or immature immune systems. It is of great significance in AIDS patients where it can cause serious morbidity through retinitis-associated blindness, and other complications, such as pneumonia and enteritis. In developed nations, it is a leading viral cause of congenital disease, where in-utero infection manifests in mental and behavioral disorders. In order to control infection and HCMV associated disease, new compounds and novel strategies must be developed. Understanding the role viral proteins play during the course of infection will help elucidate the mechanisms of HCMV pathogenesis and provide important information on potential targets for new treatments.

However, the strict species specificity of HCMV prevents any studies into the pathogenesis of the virus in an animal host. This limitation can be overcome through the use of murine cytomegalovirus (MCMV). MCMV, like HCMV, is a betaherpesvirus that exhibits similar pathogenesis in mice to HCMV infection in the human host. The genetic structure of MCMV contains significant sequence homology to HCMV AD169 in at least 78 ORFs and can thereby be used as an important tool in elucidating the functions of these ORFs in a complete in vivo system.

In our study, we have conducted a comprehensive YTH screen to identify potential interactions between approximately 170 MCMV ORFs. Growth phenotype analysis were also conducted using five different cell lines potentially involved in various aspects of CMV infection. Between these 170 predicted proteins we have identified 94 potential interactions that exhibit varying levels of essentiality depending on the type of cell infected.

We aim to understand the nature of the interactions between the viral particle and proteins encoded by the virus in order to elucidate potential mechanisms by which these proteins help to assemble and create new progeny viruses. The interactions that we have identified in this study provide a framework to predict the functions of uncharacterized viral proteins. And understanding the importance of each protein in the context of infection can further help to determine the nature of these unknown viral proteins. Together using information about known viral proteins that interact with these unknown elements, we can develop a better understanding of how all of these components contribute to viral infection which can be used to determine more effective methods to treat or prevent CMV associated diseases.

Engineered RNase P-based gene-targeting agents derived from the RNA subunit of E.coli RNase P, represent a promising approach for anti-viral applications. The focus of the research in this dissertation has been to develop anti-viral therapeutic agents from RNase P-based RNAs, with enhanced efficacy at cleaving targeted human cytomegalovirus (HCMV) mRNAs in cells. This dissertation is organized into six chapters that conclude my research. In Chapters 2 and 3, engineered RNase P-based ribozyme variants V661-IE2 and V718-A, which were generated using an in-vitro selection procedure, were designed to target HCMV mRNAs of the immediate-early protein (IE2), and the overlapping mRNA region of capsid assembly protein (AP) and protease (PR), respectively. Variant ribozyme V718-A showed a nearly 60-fold greater activity than wild type M1 RNA in cleaving target AP/PR mRNAs in-vitro. Additionally, a reduction in AP/PR expression of 98% – 99% and a viral growth inhibition of 50,000-fold was observed in cells expressing V718-A, while a 75% decrease in AP/PR expression and a viral growth reduction of 500-fold was detected in cells expressing M1-A. Variant V661-IE2 showed a nearly 50-fold greater in-vitro cleavage activity than wild type M1-IE2 in targeting IE2 mRNA. Likewise, A nearly 98% decrease in expression of IE2 and a 3,500-fold reduction in viral growth was observed in cells expressing variant V661-IE2, compared to a 75% decrease in IE2 expression and a 100-fold reduction in viral growth in cells expressing wild type ribozyme M1-IE2. These results indicate that the generated ribozyme variants selected to be highly active in-vitro are also more effective in targeting mRNAs in cultured cells, and are a potential effective approach for anti-viral therapeutic applications. In Chapter 4, engineered external guide sequence (EGS) molecules, which were generated from an in-vitro selection procedure, was designed to target mRNAs of the herpes simplex virus 1 (HSV-1) major transcriptional regulator (ICP4). The EGS variant induced human RNase P to cleave ICP4 mRNA in-vitro, with a 60-fold greater activity than EGSs generated from a natural ptRNA substrate. Additionally, a nearly 97% inhibition in ICP4 expression, and a 7,000-fold decrease in viral replication was seen in HSV-1-infected cells expressing the variant EGS, compared to a 75% reduction in ICP4 expression and a 500-fold decrease in replication in cells expressing the EGS derived form a natural ptRNA. These results suggest that engineered EGS variants can effectively block HSV-1 gene expression and viral growth in cells, and show the potential for engineered EGS variants to be developed as effective anti-viral therapeutic agents.

Attenuated strains of Salmonella bacteria represent promising nucleic acid delivery agents. Lastly, in Chapter 5 we constructed a novel strain of attenuated Salmonella for the delivery and expression of antigens hemagglutinin (HA) and neuraminidase (NA) from the H5N1 influenza virus. Here, we showed that the Salmonella vaccine induced a substantial production of anti-HA serum IgG and mucosal IgA, and anti-HA interferon-gamma producing T cells in orally-vaccinated mice. Additionally, we observed orally-vaccinated mice that expressed viral antigens HA and NA were completely protected from H5N1 virus in lethal amounts, while all mice treated with empty expression vector-carrying Salmonella were not protected. These results indicate that Salmonella-based vaccines induces strong humoral and cellular immune responses that are antigen-specific. Thus, Salmonella-based vectors can be designed to provide effective immune protection against multiple influenza virus strains. Moreover, this study highlights the potential in developing attenuated Salmonella strains as new and viable oral vaccine vectors or as effective gene-delivery agents for anti-viral gene-targeting applications in vivo.

Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause significant morbidity in neonates and immunodeficient individuals such as, AIDS patience and organ transplant recipients. With a genome size of 190-230 kilobases in size and an estimated 160 or more protein-encoding genes, HCMV is currently the largest known virus to infect humans. The functions of many of HCMV's genes remain unknown and only by understanding their role in viral infection and replication will we be able to successfully develop therapies to combat the disease.

Using a Bacterial Artificial Chromosome (BAC) containing the genome of HCMV, we constructed over 100 recombinant viruses, each with a single open reading frame (ORF) expressing its protein with an epitope tag at the C terminus end. Using these viruses, we were able to identify subcellular localization for 82 ORFs at 72 hours post infection. Our study found 17 proteins to localize to the nucleus, 51 to a juxtanuclear structure in the cytoplasm, 11 to the cytoplasm, and three to an uncharacterized structure in the cytoplasm.

We then conducted an extensive study of HCMV ORF US20, which exhibited a unique cytoplasmic localization in the previous global localization study. The US20 ORF is a member of the US12 gene family and is present only in cytomegaloviruses strains that infect rhesus monkeys, chimpanzees, and humans, suggesting that the ORF is an important factor for the infection of upper primates. We found the US20 ORF to express a 26-28kDa- seven transmembrane (TM) domain protein during the early/delayed early phase of viral replication, which localized to a cytoplasmic structure during the late phase of replication. This localization was found to be independent of late gene expression. Through immunofluorescence (IFA) studies, we found the US20 protein to be present in early endosomes but not in the ER or TGN, suggesting that the protein is present in membranes of endosomes. However, we do not believe that the protein traffics to the surface of the cell, which we showed by selective membrane permeabilization. The US20 protein was also determined to form homo-dimers, however the function of the dimerization is still unknown. Using viruses expressing truncated forms of US20, which replicated at levels similar to the wild-type, we found that deleting TM5-TM7 significantly reduced US20 protein levels, deleting TM4-TM7 abolished dimerization, and deleting TM2-TM7 destabilized the localization of US20 in the cytoplasm. Through immunoprecipitation and mass spectrometry, we found US20 to associate with valosin containing protein, sodium potassium ATPase, and succinate dehydrogenase. Currently, under the context of infection we have confirmed the interaction of US20 with valosin containing protein (VCP), which has been found to be associated with some neurodegenerative diseases. This interaction presents the hypothesis that US20 may play a role in HCMV congenital infection of the brain.

Lastly, we examined the role of a predicted SUMOylation site located near the C terminus tail of the HCMV processivity factor UL44, which forms a C clamp around the viral DNA. SUMOylation is a post-translation modification that has been found to be involved in various cellular systems, including cell cycle regulation, transcription, cellular localization, degradation, and chromatin organization. In our study, we found that mutating the lysine residue of the conserved SUMOylation motif, ψKxE, at position 410 of UL44 to an alanine did not impair viral DNA synthesis or viral replication. This result indicates that the lysine residue of UL44 is not important for replication of HCMV in fibroblasts.

The results of this dissertation bring new insight into many of HCMV's genes, specifically US20, which prior to this study had not been extensively characterized. The data I present here provides new information about US20 and generates new hypotheses relating to its function. Furthermore, my work here can serve as a foundation for the study of other genes in the US12 gene family.

Engineered M1GS ribozyme derived from RNA subunit of E. coli can be a very promising antiviral agent. The focus of this dissertation has been to develop a method for stable, safe and effective delivery of ribozyme into the viral infection sites of an animal and achieve antiviral effect. First, the M1GS expressing constructs which target the overlapping mRNA region of MCMV assembly protein (mAP) and M80 were successfully delivered into MCMV-infected CB17 SCID mice through a modified hydrodynamic transfection procedure and block viral pathogenesis. The expression of ribozymes was observed in the liver and spleen. Compared to the control groups, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SCID mice at 21 days after infection were 200 to 2,000 fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. This study successfully uses a hydrodynamic transfection method to deliver ribozymes into animal and demonstrates the feasibility of using M1GS ribozymes for inhibition of viral gene expression in animals. Second, using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, the study showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in human specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. A functional M1GS ribozyme was constructed to target the overlapping mRNA region of the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in cells that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87 - 90% in viral CSP expression and a reduction of about 5,000 fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with control groups. This study showed for the first time that ribozymes delivered via Salmonella-based vectors are highly active and specific in blocking viral infection in cultured cells. Third, a functional M1GS ribozyme that targets the overlapping mRNA region of MCMV M80.5 and protease was constructed. A novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little virulence in mice, was constructed and used for delivery of anti-MCMV ribozyme in vivo. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into targeted organs, leading to substantial expression of ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers and improved survival compared to the control group. This study provided the first direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene targeting applications in vivo.

With a genome size of 230–240 kb, human cytomegalovirus (HCMV) has the largest genome in the human herpes virus family. Once the hosts are infected with HCMV, HCMV remains latent throughout the lifetime, but the latent infection can be reactivated when the host is immunocompromised. The infection symptom is mild or subclinical diseases in immunocompetent adults, but HCMV infection leads to severe, life-threatening complications in people who are immunocompromised, including AIDS patients and transplant recipients. While there’s no vaccine available for HCMV infection, understanding the nature of HCMV replication, latency, and transmission is crucial for preventing and curing the disease.

Here we discussed the relationship between various small RNAs and HCMV. In the first part of the study, we examined one viral encoded micro RNA, HCMV-mir-UL22A*, and its influence on HCMV viral infection. We investigated the mechanism of blunt-end HCMV-mir-UL22A* inhibition of HCMV viral replication. The rapid and long lasting suppression of viral replication by blunt-end HCMV-mir-UL22A* suggests blunt-end HCMV-mir-UL22A* a promising antiviral agent. Furthermore, we studied the stagger-end HCMV-mir-UL22A* in regulating host innate immune response. Stagger-end HCMV-mir-UL22A* targets innate immune sensors and facilitate HCMV evade host innate immune response during viral infection. Blunt-end HCMV-mir-UL22A* showed potential for treating CMV infection, while stagger-end HCMV-mir-UL22A* helped us understand HCMV viral innate immune evasion strategy.

In the second part of the study, we focused on applying RNA based precise gene editing technology to treat HCMV infection. Curing CMV infection requires disruption of the viral genome by novel gene editing technology. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is a gene editing tool for the site-specific cleavage of DNA. We adapted CRISPR/Cas9 system in CMV infected cells. CRISPR/Cas9 could suppress HCMV and MCMV infection in vitro in cell model. Our study suggests CRISPR/Cas9 can potentially be applied to treat CMV infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with several human malignancies including the cancers Kaposi’s sarcoma and primary effusion lymphoma. As with other herpesviruses, KSHV can enter either latency or undergo lytic replication within its host. Several latency-associated open reading frames (ORF) and a few lytic cycle-associated ORFs are relatively well-studied. However, the majority of the 140.5kb long unique region of the genome, which contains about 90 ORFs, is largely uncharacterized outside of in silico studies. We took advantage of the BAC16, a bacterial artificial chromosome clone of a KSHV genome, as well as the iSLK cell line, a permissive cell line with tight latency when infected and efficient reactivation upon chemical induction, in order to generate targeted KSHV mutants to better understand the functionality of KSHV ORFs.

In chapter 2, I explain how we generated 90 mutants using the BAC16, each characterized by the disruption of a single ORF. Mutant BACs were introduced into iSLK cells by transfection and classified as essential or nonessential for the maintenance of latency in this cell model. Then transfected cells were chemically induced into lytic replication and the amount of infectious virus generated and released into the supernatant was quantified. This allowed us to classify ORFs as essential or nonessential for reactivation and infectious virus generation. Notably, we identified several core herpesvirus ORFs as non-essential which had previously been classified as essential in other herpesviruses, including ORF 17, 42, 57, 64, 66, 67, 69, glycoprotein L, glycoprotein H, and glycoprotein B.

I then further characterized the nonessential mutants by using growth curve assays under conditions favoring latency, reactivation, and lytic replication in chapter 3. This allowed me to isolate the roles of KSHV ORFs to specific viral life cycle stages, and to take a high-level approach of understanding which ORFs disrupt or promote infectious virus generation under these conditions. We found that ORFs 16, 46, 58, 60, 61, and 67 are deficient in virus generation under lytic conditions and likely play an important role in the early stages of viral infection to prime the virus for successful reactivation. We also identified several KSHV-specific ORFs (K3, K4, and K5), which function to inhibit reactivation. Finally, we discovered that ORFs 49, 50, 56, and K11 promote spontaneous reactivation during latency while ORFs 11AA, 72, K6, and K7 repress it, underscoring the biological importance of viral control of this phenomenon, which plays a large role in pathogenesis and is not yet fully understood. In chapter 4, I further dissected the effects of ORF disruption on the viral life cycle. Here we monitored lytic antigen expression of BAC16 or mutant infected cells by measuring the percentage of infected cells expressing immediate-early (ORF45), early (K8α), or late (K8.1A/B) lytic antigens under conditions favoring latency or reactivation. We found that ORF20 tempers lytic gene expression upon reactivation. Additionally, ORF 10.1, 20, 23, 27, 58, and 67 seem to facilitate the burst of lytic gene expression shortly after infection, while ORF 11, 28, K10, K10.5, and K9 promote the establishment of latency after this.

Human Cytomegalovirus (HCMV) is a ubiquitous virus that is a leading cause of both congenital infections in neonates and opportunistic infections in immunodeficient persons such as HIV/AIDS patients and solid-organ transplant recipients. Few human viruses compare to the complexity, broad cell tropism, and life-long persistence observed in HCMV infections. HCMV has one of the largest genomes of any known human viral pathogen with an approximate size of 230kbp of DNA that encodes roughly 175 canonical genes. The genes encoded by HCMV have been characterized by forward genetics using a single-gene deletion approach to understand the essential or dispensable nature of each HCMV gene necessary for viral replication in vitro, however the precise biological functions of most HCMV genes remain incompletely understood. HCMV encodes an unparalleled number of proteins that subvert both innate and adaptive immunity, yet currently the role of the human complement system in HCMV infection and immunity remain poorly understood even though this important branch of innate immunity has been well characterized in other related human herpesviruses.

Previous studies have reported that HCMV virions incubated in serum (a source of complement) are not neutralized but are nevertheless coated with activated complement proteins that normally proceed the direct lysis of microorganisms. This observation suggests that HCMV virions not only activate one or more pathways of complement, but are able to inhibit complement-mediated neutralization perhaps by a viral encoded complement regulating protein. In this dissertation, we screened 6 human complement proteins (CD55, Factor H, Mannose Binding Lectin 2, Mannan-Associated Serine Protease-1, Properdin, and C1q Binding Protein) against an HCMV genomic library in yeast containing 167 viral open reading frames. Out of 1,002 possible HCMV-Complement interactions tested, we identified 121 (8.2%) positive-protein interactions by yeast-two-hybrid among all six complement proteins tested and validated a subset of these novel HCMV-Complement interactions in human cells by co-immunoprecipitation. To date, our study is the largest, most comprehensive, and systematic investigation of interactions between HCMV the complement system, and provides a framework to further investigate HCMV-Complement interactions that may underlie undiscovered mechanisms of innate immune evasion by HCMV and potentially inform the development of novel drugs and vaccines for the treatment and prevention of HCMV infections.

During our studies, a novel coronavirus known as Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) emerged and spread rapidly in humans, which led to a pandemic and significant loss of life globally. In response to the pandemic, SARS-CoV-2 vaccines were developed but their efficacy has been undermined by the increasing emergence of SARS-CoV-2 isolates with mutations in the Spike (S) protein which is the primary vaccine antigen in all licensed SARS-CoV-2 vaccines. These observations underscore the significant need to identify additional antigens for possible inclusion in the next-generation of SARS-CoV-2 vaccines. To address this significant unmet medical need, we investigated the vaccine potential of a highly conserved 30 amino acid transmembrane domain epitope of the SARS-CoV-2 Envelope (E) protein in mice. To increase immunogenicity, we conjugated the E-protein transmembrane domain to the immunogenic carrier protein Keyhole Limpet Hemocyanin (KLH) to generate a vaccine antigen herein referred to as KLH-E. Mice were immunized with one or two doses of KLH-E vaccine or aluminum control vaccine intramuscularly in thirty-day intervals. The KLH-E vaccine elicited serum IgG antibodies and antigen-specific T-cells to each subunit of the vaccine (KLH and E), and to our surprise induced anti-E IgG antibodies and T-cells that recognize the native E protein from the genetically divergent but related human coronavirus 229E (HCoV-229E). Furthermore, pooled sera from mice had neutralizing activity against SARS-CoV-2 pseudovirions expressing S proteins from emerging variants of concern (Beta, Delta, Omicron, XBB), and had neutralizing activity against HCoV-229E which shares only a modest 32% E protein sequence homology to the vaccine antigen. Taken together, these results suggest that the SARS-CoV-2 E protein transmembrane domain contains important epitopes that are conserved across different human coronaviruses and demonstrates that immunization with the E protein transmembrane domain from SARS-CoV-2 can induce cross-reactive immune responses that may confer protection against emerging SARS-CoV-2 variants and other genetically diverse human coronaviruses.

Kaposi Sarcoma-associated herpesvirus (KSHV) is a medically important virus that causes two types of human cancer. Infection with KSHV can result in viral persistence for the lifetime of the host. Viral persistence, commonly referred to as latency, is characterized by the absence of gene expression. Reactivation from latency results in entry into the lytic cycle which is characterized by the full repertoire of viral gene expression. A key pathogenic step in KSHV-mediated disease is viral reactivation and viral lytic gene expression. Immunosuppression, regardless of the source (HIV-mediated or iatrogenic), results in log-fold increases in the risk of developing KSHV-mediated diseases.We adopted a tractable system to study a KSHV genome derived from a clinical isolate. This KSHV genome has been engineered for stable propagation in E. coli. The strain of E. coli harboring the KSHV genome has been engineered for the use of a scarless, site-directed mutagenesis procedure. Parental and mutagenized virus can be introduced into a human cell line via transfection. Transfection of parental virus into this cell line results in stable latency. This cell line has also been engineered to express the major viral-transactivator under an inducible promoter. Expression of the viral-transactivator is sufficient to reactivate KSHV from latency, drive the full repertoire of viral gene expression, and results in significant cell-free progeny. We implemented this experimental workflow to mutagenize all known canonical Open Reading Frames (ORFs) and several other uncanonical ORFs. Transfection and cell-free progeny analyses resulted in the identification of ORFs essential for completion of the viral lifecycle. Growth analyses of non-essential mutants resulted in the identification of ORFs important for induced and spontaneous viral reactivation. Phenotypic screening of essential mutants using light and electron microscopy resulted in the identification of ORFs playing vital roles in viral lytic gene expression and capsid formation and maturation. The results of my thesis reveal the identification of novel antiviral targets and live-attenuated vaccine candidates. Moreover, these data suggest a new model for KSHV latency and reactivation. This model proposes that KSHV actively communicates with and responds to the host’s immune system in a concerted effort to fine-tune viral latency and reactivation. Supplementary analyses, not provided herein, are underway to elucidate the molecular details for discrete steps in viral egress.