Retroviral infection requires an initial interaction between the host cell and the virion. This interaction is predominantly mediated by an envelope (env) protein exposed on the external face of the virion. For gammaretroviruses, such as feline leukemia virus (FeLV), the receptor-binding domain (RBD) is located in the N terminus of env. The RBD forms a distinct domain that is sufficient for binding to the host receptor, but is inefficient in the absence of the corresponding C terminal env, Cdomain, sequence in viral infection studies. I developed a series of hybrid constructs between subgroup C, A and T FeLVs that use distinct receptors for infection to determine the role of Cdom in FeLV binding and infection. Using this approach, I have shown that the C domain (Cdom) of FeLV-C env forms a second receptor-binding domain, distinct from its RBD, which is critical for efficient binding and infection of FeLV-C to host cells expressing FLVCR1. I propose that this mechanism of interaction is conserved for all gammaretroviruses. My results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells. Upon infection with FeLV-C virus, cats develop a disease known as pure red cell aplasia (PRCA). This disease is characterized by a defect in erythropoeisis that results in a decreased number of mature erythroid cells. PRCA has been suggested to be caused by the FeLV-C env binding to and disrupting the host receptor, FLVCR1. Interestingly, feline PRCA is clinically identical to Diamond Blackfan Anemia (DBA), a fatal congenital anemia characterized by a specific disruption in erythroid progenitor cellular development. I show that erythroid cells from five DBA patients exhibit low levels of total FLVCR1 transcript expression. In addition, the DBA patients express unique alternatively spliced FLVCR1 transcripts. These alternatively spliced transcripts encode FLVCR1 proteins that are defective in their cellular expression, cell surface localization, and receptor function. Taken together, I propose that the specific anemia observed in DBA is caused by decreased levels of functional FLVCR1 protein due to lowered and alternative splicing of FLVCR1 transcript.

Retroviral infection requires an initial interaction between the host cell and the virion. This interaction is predominantly mediated by an envelope (env) protein exposed on the external face of the virion. For gammaretroviruses, such as feline leukemia virus (FeLV), the receptor-binding domain (RBD) is located in the N terminus of env. The RBD forms a distinct domain that is sufficient for binding to the host receptor, but is inefficient in the absence of the corresponding C terminal env, Cdomain, sequence in viral infection studies. I developed a series of hybrid constructs between subgroup C, A and T FeLVs that use distinct receptors for infection to determine the role of Cdom in FeLV binding and infection. Using this approach, I have shown that the C domain (Cdom) of FeLV-C env forms a second receptor-binding domain, distinct from its RBD, which is critical for efficient binding and infection of FeLV-C to host cells expressing FLVCR1. I propose that this mechanism of interaction is conserved for all gammaretroviruses. My results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells. Upon infection with FeLV-C virus, cats develop a disease known as pure red cell aplasia (PRCA). This disease is characterized by a defect in erythropoeisis that results in a decreased number of mature erythroid cells. PRCA has been suggested to be caused by the FeLV-C env binding to and disrupting the host receptor, FLVCR1. Interestingly, feline PRCA is clinically identical to Diamond Blackfan Anemia (DBA), a fatal congenital anemia characterized by a specific disruption in erythroid progenitor cellular development. I show that erythroid cells from five DBA patients exhibit low levels of total FLVCR1 transcript expression. In addition, the DBA patients express unique alternatively spliced FLVCR1 transcripts. These alternatively spliced transcripts encode FLVCR1 proteins that are defective in their cellular expression, cell surface localization, and receptor function. Taken together, I propose that the specific anemia observed in DBA is caused by decreased levels of functional FLVCR1 protein due to lowered and alternative splicing of FLVCR1 transcript.

The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.

Lucerne transient streak sobemovirus (LTSV) supports the replication of a small, 322-nucleotide, untranslated virusoid (vLTSV) that has an extensively base-paired, viroid-like structure. Since vLTSV does not code for its own proteins or share sequence homology with its helper virus (LTSV), it is presumed that it uses structural motifs to signal the helper virus (and host) machinery for its replication. In order to elucidate these structural domains, insertion-deletion mutations were introduced to disrupt the secondary structure. Infectivity assays of these mutants showed that they were all lethal, except a 9-nucleotide, palindromic insertion, which preserved the overall rod-like structure of the virusoid. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed that the palindromic sequence was replicated up to twelve days of infection but discarded afterwards. Results indicate that vLTSV has an optimum size and secondary structure for replication and packaging within the LTSV helper virus.

Lucerne transient streak sobemovirus (LTSV) supports the replication of a small, 322-nucleotide, untranslated virusoid (vLTSV) that has an extensively base-paired, viroid-like structure. Since vLTSV does not code for its own proteins or share sequence homology with its helper virus (LTSV), it is presumed that it uses structural motifs to signal the helper virus (and host) machinery for its replication. In order to elucidate these structural domains, insertion-deletion mutations were introduced to disrupt the secondary structure. Infectivity assays of these mutants showed that they were all lethal, except a 9-nucleotide, palindromic insertion, which preserved the overall rod-like structure of the virusoid. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed that the palindromic sequence was replicated up to twelve days of infection but discarded afterwards. Results indicate that vLTSV has an optimum size and secondary structure for replication and packaging within the LTSV helper virus.

The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.

Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviruses are enveloped, rod shaped viruses with circular, double-stranded DNA genomes. These viruses infect arthropods, primarily in the order Lepidoptera, although members of this virus family also infect species of Diptera, Hymenoptera, and Crustacea. The majority of these viruses undergo a bi-phasic cycle with one phase defined by the production of a budded virus (BV) form, responsible for cell to cell transmission, and the other defined by the production of an occlusion-derived virus (ODV) form, responsible for host to host transmission. The proto-typical member of the Baculoviridae family is considered to be Autographa californicaM Nucleopolyhedrovirus (AcMNPV). Its 133,894 base pair genome is predicted to encode for 156 proteins, a large number of which are essential for virus replication.. In this current work, we have further characterized two viral proteins that are highly conserved among baculoviruses. The first of these is an ortholog of the fibroblast growth factor family of proteins with sequence homology to the Drosophila Branchless protein as well as the mammalian FGF- 9, -16 and -20 subfamily. Despite its high degree of conservation among baculoviruses, the viral fibroblast growth factor (vFGF) is considered a non-essential protein, although its deletion from the genome does affect the lethality of the virus when ingested per os. In our study, we were able to localize vFGF to the membrane of BV. Its presence on the envelope affected the ability of the virus particle to bind to both heparin in vitro and to the cell surface in vivo, and may play a role in the attachment phase prior to virus entry. We also characterized AcMNPV’s open reading frame 109 (Ac-orf109). Unlike vFGF, Ac-orf109 is essential for virus replication since its deletion results in a complete lack of BV production. Transmission electron microscopy of cells transfected with an Ac-orf109 deletion virus shows the full range of virus-associated structures including mature capsid formation but there appears to be a deficiency in capsid egress out of the nucleus. Furthermore, the ODV retained in the nucleus appear to lack microvesicular membranes, an essential component for host to host transmission of infection.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

Feline leukemia viruses (FeLVs) are pathogenic retroviruses of the domestic cat. FeLV transmission and emergence of pathogenic variants show striking similarity to HIV pathogenesis. The emergence of pathogenic subgroup-C FeLV from the transmitted subgroup-A FeLV coincides with a switch in host receptor used for infection as a result of mutations in the viral envelope protein (Env). I have characterized a novel FeLV Env that may represent an evolutionary intermediate between FeLV-A and FeLV-C. I have also reported evidence suggesting that FeLVs may use co-factors/co-receptors for infection. I have found that FeLVs inefficiently infect murine NIH3T3 cells overexpressing FeLV receptors (NIH3T3/Receptor). I have provided evidence that the low infection is caused by a block at a post-binding but pre-entry stage of FeLV infection. Furthermore, fusion of NIH3T3/Receptor cells with highly susceptible cells rescues inhibition to infection suggesting that FeLVs, like HIV, may also use co-receptors for infection.