<p> Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the known human cancer viruses, causing Kaposi's sarcoma and primary effusion lymphoma in immunosuppressed patients. Although of medical concern, the mechanisms through which the virus causes cancer remain poorly understood. Researchers speculate that the lytic phase contributes to the development of human cancers by this virus.</p><p> KSHV, like other herpesviruses, is predominantly latent in the human host, but undergoes lytic activation to produce infectious viral particles. In the process, the virus hijacks the host machinery to express large quantities of viral genes via a process known as the host shutoff effect. The virus then replicates its DNA and assembles viral capsids within nuclear viral replication compartments. Viral proteins act in various locations within the cell depending on their function. However little is known about the location of viral transcripts and how their localization relates to their function. Thus I sought to understand the localization of viral transcripts to gain insight into the spatiotemporal regulation of the lytic phase.</p><p> Using fluorescence in situ hybridization (FISH) and immunofluorescence (IF), I observed that particular viral transcripts accumulate within the nucleus in or near replication compartments. This occurs late in the lytic phase coinciding with viral DNA replication. My findings indicate that the mechanism is independent of the host shutoff effect and splicing, but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, polyadenylated nuclear (PAN) RNA. PAN RNA is essential for the viral life cycle and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.</p><p> One key regulator of the KSHV lifecycle is a long noncoding RNA (IncRNA) called the polyadenylated nuclear (PAN) RNA. PAN RNA is an early gene product comprising nearly 80% of total polyadenylated cellular transcripts in lytic infected cells. Studies on its function demonstrate that PAN RNA is a regulator of virion production through modulation of viral genes.</p><p> A glimpse into the mechanism comes from recent <u>ch</u>romatin isolation by RNA purification (CHIRP) studies on lytic KSHV-infected B lymphocytes. The studies revealed widespread binding of PAN RNA to viral and host chromatin, but could not identify an underlying mechanism. The most feasible approach to study function is a genetic knockout. However, a complete PAN RNA gene deletion is unachievable in the KSHV genome due to an overlapping open reading frame, K7. In a related gammaherpesvirus, rhesus rhadinovirus (RRV), a computational search uncovered a PAN RNA homologue, whose sequence does not overlap with any known genes. I found that RRV PAN RNA is present at about 150,000 copies per cell and organized the purchase of RRV &Delta;PAN RNA constructs to facilitate study of PAN RNA's mechanism.</p><p> Capitalizing on the RRV homolog, Dr. Johanna B. Withers and I compared changes in chromatin association by PAN RNA between homologs and over the lytic phase with CHART (<u>c</u>apture <u>h</u>ybridization <u> a</u>nalysis of <u>R</u>NA targets). After careful analysis, the data suggest that chromatin-association by PAN RNA is nonspecific and that the mechanism of regulation by PAN RNA is not primarily related to chromatin remodeling. With this is mind, I looked to another potential mechanism, one related to binding by nuclear relocalized cytoplasmic polyAbinding protein (PABPC). </p><p> Both PAN RNA homologs associate with several host proteins, one of which is cytoplasmic polyA-binding protein (PABPC). Upon lytic induction, SOX, the host shutoff mediator, facilitates degradation of messages in the cytoplasm, causing the PABPC to relocalize to the nucleus. Nuclear relocalized PABPC binds KSHV PAN RNA at ~8-10 proteins per RNA molecule. I hypothesize that a function of PAN RNA is to act as nuclear relocalized PABPC sponge to facilitate preferential expression of viral genes and assembly of virions.</p><p> I designed mutant to capitalize on a unique feature of PAN RNA, the triple helical stabilization element (ENE). At the 3' end a triple helix (ENE) forms with polyA tail, protecting PAN RNA from deadenylation, stabilizing it. I reduced the length of the polyA-tail to eight adenylates, which permits formation of the triple helix, but falls below the 20-adenylate footprint of PABPC. The RRV PAN constructs would supplement RRV &Delta;PAN RNA virus to examine if the tailless PAN RNA mutants rescue the loss of virion production seen previously during the downregulation of KSHV PAN RNA. The results from these experiments would yield a deeper understanding of host-virus interactions and will provide insights into the importance of PABP-binding for the function of a nuclear noncoding RNA.</p><p>

Thesis (Ph.D.)--Syracuse University, 2008. / "Publication number: AAT 3323047."

<p> In order to initiate an infection, bacteriophages must deliver their large, hydrophilic genomes across their host&rsquo;s hydrophobic cell wall. Bacteriophage &phiv;X174 accomplishes this task with a set of identical DNA piloting proteins. The structure of the piloting protein&rsquo;s central domain was solved to 2.4 &Aring; resolution. In it, ten proteins are oligomerized into an &alpha;-helical barrel, or tube, that is long enough to span the host&rsquo;s cell wall and wide enough for the circular, ssDNA to pass through. This structure was used as a guide to explore the mechanics of &phiv;X174 genome delivery. In the first study, the H-tube&rsquo;s highly repetitive primary and quaternary structure made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein&rsquo;s three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. </p><p> The tube&rsquo;s inner surface was altered in the second study. The surface is primarily lined with amide and guanidinium containing amino acid side chains with the exception of four sites near the tube&rsquo;s C-terminal end. The four sites are conserved across microvirus clades, suggesting that they may play an important role during genome delivery. To test this hypothesis and explore the general role of the amide and guanidinium containing side chains, the amino acids at these sites were changed to glutamine. The resulting mutants had a cold-sensitive phenotype at 22 &deg;C. Viral lifecycle steps were assayed in order to determine which step was disrupted by the mutant glutamine residues. The results support a model in which a balance of forces governs genome delivery: potential energy provided by the densely packaged viral genome and/or an osmotic gradient push the genome into the cell, while the tube&rsquo;s inward facing residues exert a frictional force on the genome as it passes. </p><p> Bacteriophage must first identify a susceptible host prior to genome delivery. In the final study, biochemical and genetic analyses were conducted with two closely related bacteriophages, &alpha;3 and ST-1. Despite ~90% amino acid identity, the natural host of &alpha;3 is <i>Escherichia coli</i> C, whereas ST-1 is a K-12-specific phage. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: mutant progeny from co-infected cells did not display the phenotype. Novel virus propagation and selection protocols were developed to isolate host range expansion mutants. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.</p><p>

<p> The molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.</p><p>

<p> The discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast <i> Saccharomyces cerevisiae</i>. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an <i>in vitro </i> yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share. </p><p>

Dengue Fever is a global problem with a worldwide effectiveness that put 2.5 Billion people under the risk, infect 50 million people and causes 30000-50000 people death each year. DHF was first recognized in the 1950s during the dengue epidemics in the Philippines and Thailand. By 1970 nine countries had experienced epidemic DHF and now, the number has increased more than fourfold and continues to rise. Today emerging DHF cases are causing increased dengue epidemics in the Americas, and in Asia, where all four dengue viruses are endemic. Vaccine development against Dengue Virus has been impossible to date, due to effective vaccination to prevent DHF will require a tetravalent vaccine, because epidemiologic studies have shown that preexisting heterotypic dengue antibody is a risk factor for DHF a lethal form of dengue fever. To date there are no pharmaceutical treatments for Dengue fever. DV proteome is composed of 8 proteins and dengue virus protease is one of them and it is essential for virus replication therefore it has being a potential drug target for dengue fever treatment. Active-site inhibitors of proteases have been successfully used to treat other virally transmitted diseases of global importance such as HIV and Hepatitis C, however protease active site inhibitors they are subject to development of resistance. In addition, it is often difficult to target the active site due to overlapping sequence preference with endogenous human proteases. This overlap in specificity of active-site inhibitors contributes to unwanted side effects. A principal bottleneck is that traditional drugs are designed to bind to one protein and control the function of only that protein at the active (primary functional) site. Unfortunately, similar active sites are almost always present in related proteins, leading to lack of drug specificity and thus to many unwanted side effects. A promising alternative is to use allosteric sites. Allosteric sites are cryptic drug binding sites that are spatially distinct from the active site. They allow the protein to be locked into a unique conformation that either turns the protein `on' or `off'. Historically it has been difficult or impossible to find allosteric sites, because mechanisms of allostery were poorly understood and tools for their identification were lacking. Our unique combination of experimental approaches has enabled us to identify new allosteric sites and new allosteric mechanisms of control in four different biomedically important proteins. Many proteins have multiple allosteric sites. Allosteric sites can often be exploited for much more specificity than active sites. We have developed and implemented a technology for discovering new allosteric sites in proteases and have applied this to NS2B-NS3pro from dengue virus type 2. In this thesis we report an allosteric site in NS2B-NS3pro that can be targeted in biomedical studies. We also have shown that the internal flexibility of NS2b-NS3pro is critical for catalytic activity.

Viruses have evolved to exploit cellular pathways and machinery in order to deliver their genome to the cell, replicate, and produce viral progeny. Nonenveloped viruses must overcome membrane barriers to infect host cells and trigger lysis for virion release. The model nonenveloped virus, Simian Virus 40 (SV40), is bound at the cell surface and eventually delivered to the endoplasmic reticulum (ER) where penetration occurs resulting in delivery of the viral genome to the nucleus by an unknown mechanism. During the later stages of infection viral progeny are assembled in the nucleus and are liberated from the host cell through a cytolytic process. SV40 appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection for virus release. Bacterially expressed and purified VP4 forms size selective pores in membranes. To investigate the role of VP4 in host cell lysis an inducible expression system was used to produce VP4 in mammalian cells. The viral protein was mainly localized along the nuclear envelope and correlated with the mislocalization of nuclear proteins and was associated with cell death. These results indicate that VP4 acts as a viroporin in the nuclear membrane to promote virus release. Previous results indicated that the two minor structural proteins, VP2 and VP3, may act as membrane proteins during viral infection. Studies using purified proteins, bioinformatics, a cell-free membrane insertion assay and a thorough examination of viral propagation, assembly and infection processes have provided new insights into the role of the minor structural proteins during infection. Targeted disruption of the viroporin activity of VP2 and VP3 inhibited viral infection. Together, these results support that the late viral proteins VP2, VP3 and VP4 each act as viroporins and serve as critical triggers for the progression of the viral life cycle. This investigation provides new insight into how the viroporin activity of the late viral proteins is utilized in viral infection and release.

Carbohydrates represent a keystone among biological molecules. Well known as a source of energy, sugars also form the backbone of various biopolymers, act as markers and receptors for cellular communication and modulate lipid and protein functions. As such a powerful class, carbohydrates represent a useful pool from which both nature and man have drawn structures to produce biologically active compounds with a variety of modes of action. Beyond their importance to biology, sugars have represented attractive synthetic targets to chemists given their densely functionalized scaffolds. The work presented in this thesis aims to employ synthetic chemistry to provide both natural and designed carbohydrates in order to carry out biological studies to improve our understanding of these compounds' particular effects. In the first part, a synthesis is developed for the carboline disaccharide domain of the cytotoxic enediyne, shishijimicin A. The route employs a Reetz-Müller-Starke reaction to install the domain's quaternary center, with addition of a carboline dianion to complete the target. Iminosugars represent the focus of the second portion of the thesis. These polyhydroxylated alkaloids have long been investigated for their ability to mimic single sugars, inhibiting various glycosidases and glycosyltransferases. The endocyclic nitrogen atom of members of this class can act as a functional handle for alkylation, with increased chain length increasing both potency of enzyme inhibition and toxicity in cellula. Specific iminopyranose structures with D-gluco stereochemistry have broad-spectrum antiviral activity, while those with D-galacto stereochemistry are antiviral with respect to hepatitis C, but not other genetically related viruses. Reported herein are syntheses of classes of iminosugars to determine the influence of both N-alkylation chain length and iminopyranose stereochemistry on the spectrum of antiviral activity. Complementing antiviral activity with isolated enzyme inhibition assays, the work aims to identify new targets for next generation antivirals. Finally, the prototypical iminosugar, D-deoxynojirimycin, is conjugated to a second natural product, D-&alpha;-tocopherol. By replacing the more common normal alkyl group with a lipid, the goal was to reduce cellular toxicity, while also taking advantage of the natural active transport for the lipid to increase uptake of the drug. Surprisingly, this change provided a marked shift in selectivity of enzyme inhibition and antiviral ability. In order to fully characterize the mechanism, the mentioned enzymatic and antiviral studies were supplemented with lipidomic, STED-microscopy and pharmacokinetic investigations.