Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-10, Section: B, page: 6531. Adviser: Donald Ganem.

Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0768. Adviser: Robert W. Mahley. Includes supplementary digital materials.

Thesis (Ph.D.)--Indiana University, Dept. of Chemistry, 2009. / Title from PDF t.p. (viewed on Jul 19, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7590. Adviser: Peter J. Ortoleva.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3961. Adviser: Daniel W. Pack. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Mumps virus is member of the paramyxovirus family of viruses. The Urabe strain of mumps was attenuated to produce a vaccine that was introduced in several countries, but withdrawn from the market when it was discovered to be insufficiently attenuated. Two vaccine-associated Urabe viruses have been shown to have different tropisms in tissue culture. Gw7, isolated directly from the vaccine and which has been shown to be attenuated in an animal model, shows limited growth in human lung epithelial cells (A549) compared to monkey kidney cell line (Vero). 1004 10/2 is a virus isolated from a patient with post-vaccination meningitis caused by the Urabe vaccine and this virus grows to high titres in both A549 and Vero cells. A limited number of genetic differences between Gw7 and 1004 10/2 have been identified that must account for the growth differences in tissue culture. Using a combination of functional assays and genetic analysis of other Urabe viruses with growth patterns similar to Gw7 or 1004 10/2, it was demonstrated that the failure of Gw7 to replicate well in A549 cells is not due to differences in the sensitivity to interferon, differences in receptor binding, the M protein, or the SH protein. The results implicated the L protein. This was further supported by the observations of reduced protein synthesis and RNA synthesis in A549 cells compared to Vero cells. This suggested that genetic differences between the viruses caused differences in the function of the L protein during transcription of mRNA, which resulted in the observed tropisms.

During viral infection, the host subjects the virus to an array of protective mechanisms. Viruses have evolved evasion strategies to thwart these defenses, and must simultaneously regulate multiple cellular pathways and resources to achieve successful replication. To orchestrate such complex regulation, viruses, including herpes simplex virus 1 (HSV-1), rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein counteracts various host defenses by targeting cellular factors for degradation. We undertook a proteomic screen to identify binding partners of ICP0, and identified the Tripartite Motif 27 (TRIM27) protein, a cellular transcriptional repressor, as a novel interacting protein of ICP0. This interaction resulted in rapid loss of TRIM27 during HSV-1 infection. However, replication of an ICP0-null mutant virus required TRIM27, suggesting a complex interaction between TRIM27 and viral infection. To further characterize regulation of TRIM27 by HSV-1, we evaluated whether infection affected TRIM27 levels independently of ICP0. Infection with an ICP0-null virus resulted in TRIM27 protein loss, but at a greatly reduced rate. TRIM27 protein exhibited a short half-life in uninfected cells, indicating that viral regulation of transcript levels could affect protein levels during infection. HSV-1 reduced TRIM27 transcripts through the virion host shutoff (VHS) function and a global inhibition of host transcription. The compound regulation of TRIM27 levels during infection demonstrated the redundant mechanisms by which HSV-1 regulates the cellular proteome. Because degradation targets of ICP0 often function to restrict viral infection, we hypothesized that TRIM27 could contribute to an antiviral pathway. Based on the involvement of TRIM27 in programmed cell death resulting from Tumor Necrosis Factor (TNF) signaling, we evaluated the role of TRIM27 in cells treated with TNF. TRIM27 was required for TNF-dependent programmed necrosis, or necroptosis, in mouse cells. HSV-1 infection of these cells induced TNF-dependent necroptosis, reducing viral yield. This pathway required mouse TRIM27, and cells expressing human TRIM27 in place of mouse TRIM27 did not exhibit necroptosis. The differing capacities of mouse and human TRIM27 to support necroptosis during HSV-1 infection suggested that TRIM27 contributes to species-specific restriction of HSV-1 and to the selective pressure driving viral evasion of this protective host response. / Medical Sciences

The host response to the family of nuclear replicating DNA viruses or the herpesviruses includes the intrinsic, innate and adaptive arms of the immune system. Intrinsic resistance is a constitutively active line of defense against virus infections and members of the Tripartite Motif (TRIM) superfamily of proteins; such as TRIM5 and TRIM19/PML in nuclear domain 10 (ND10) bodies are important restriction factors in this system. Nuclear intrinsic restriction against the prototypical DNA virus, herpes simplex virus 1 (HSV-1) includes interferon-inducible protein 16 (IFI16) and ND10 bodies. However, the viral E3 ubiquitin ligase, ICP0, encoded by wild-type HSV-1, targets these intrinsic immune proteins for degradation. Previous reports on the anti-viral function of TRIM22, the human paralog of the prototypical TRIM5α protein, emphasized its role as a gene of the innate immune system, particularly its expression as a Type I and Type II interferon-stimulated gene and its antiviral function against retroviruses. This study shows that TRIM22 has an additional intrinsic immune role against DNA viruses, using the herpesviruses as an example of a family of DNA viruses. We report that TRIM22 is a novel restriction factor of HSV-1 and limits HSV-1 ICP0-null virus replication. The TRIM22-mediated restriction of HSV-1 occurs after nuclear entry but prior to viral immediate-early gene transcription, by promoting histone occupancy and heterochromatinization to reduce immediate-early viral gene expression. The ICP0-rescued virus evades the TRIM22-specific restriction by a mechanism independent of TRIM22 degradation. We also demonstrate that TRIM22 inhibits other DNA viruses, including representative members of the β- and γ- herpesviruses. These results collectively show that TRIM22 acts in the nucleus, and provide evidence that TRIM22 restricts HSV gene expression by promoting histone occupancy on the viral genes. Furthermore, we identified seven haplotypic variants of TRIM22 and propose that amino acid substitutions in the linker L2 domain and the coiled-coil domain of TRIM22 alter the magnitude of its restriction against the herpesviruses. Together, these results argue for the importance of the TRIM22 gene as a restriction factor against herpesviruses and offer a novel avenue for further investigation on the role of TRIM genes in host genetic variation in herpesviral susceptibility. / Medical Sciences

RIG-I is a key cytosolic sensor of many RNA viruses, including dengue virus (DV), the most significant arboviral pathogen. Upon viral RNA binding, RIG-I signals via the adaptor protein MAVS, located at mitochondria, to induce the expression of interferons (IFNs), proinflammatory cytokines and interferon-stimulated genes (ISGs), thereby establishing an antiviral state. Among the ISGs, the IFITM proteins are critical for antiviral restriction of numerous pathogenic viruses including DV by inhibiting viral entry. Here, we uncover how DV escapes RIG-I-mediated immunity. The NS3 protein of DV binds directly to 14-3-3ε, a mitochondrial-targeting protein that is essential for translocation of RIG-I from the cytosol to mitochondria. Specifically, NS3 blocks 14-3-3ε from forming a “translocon” complex with RIG-I and its upstream activator, TRIM25, thereby inhibiting RIG-I translocation to mitochondria for MAVS interaction and antiviral signaling. Furthermore, RIG-I that fails to translocate to mitochondria is degraded in a lysosome-dependent manner in DV- infected cells. Intriguingly, NS3 binds to 14-3-3ε using a phosphomimetic motif that resembles a canonical phospho-serine/threonine motif found in cellular 14-3-3-interaction partners. We engineer a recombinant DV encoding a mutant NS3 protein deficient in 14-3-3ε binding (DV2KIKP) and find that this mutant virus is attenuated in replication compared to the parental virus. Strikingly, DV2KIKP fails to antagonize RIG-I and elicits high levels of IFNs, proinflammatory cytokines and ISGs in human hepatocytes and monocytes. Taken together, our data reveal a novel phosphomimetic-based mechanism for viral antagonism of innate immunity and provide a foundation for DV vaccine development. DV can infect cells directly, or complex with non-neutralizing antibodies to infect Fc- receptor-bearing cells in a secondary infection, which is associated with severe disease. While it has been shown that IFITMs restrict DV direct infection, it is unknown if the latter process, commonly termed antibody-dependent enhancement (ADE), might bypass IFITM-mediated restriction. Comparison of direct and ADE-mediated DV infection shows that IFITM proteins restrict both infection modes equally, suggesting that upregulation of IFITMs may be a therapeutic strategy. In summary, our work elucidates several molecular aspects of the interplay of DV with the human immune response, which may guide the rational design of vaccines and antivirals. / Medical Sciences

The incredible flexibility and specificity of the humoral immune response is dependent on the highly diverse repertoire of naïve and affinity-matured antibodies. Utilizing and understanding the power of this response requires high-throughput approaches. This thesis describes three projects that use recent advances in DNA sequencing and synthesis to develop and apply methods to probe the diversity of these responses at unprecedented depth. Chapter 2 describes a synthetic antibody library designed for high-throughput sequencing assisted selection which enables rapid in vitro selection of antibodies that bind specifically to a target of interest by bypassing the need for laborious single-clone screening for specific binding. Chapter 3 describes a high-throughput assay for detection of antibodies against all known human viruses using immunoprecipitation and high-throughput sequencing of bacteriophage displaying a library of peptides tiling through the proteome of all known human viruses. And last, chapter 4 describes the use of immunoprecipitation and high-throughput sequencing of both bacteriophage displayed peptides from the human peptidome and ribosome displayed proteins from the human proteome to identify a novel subclass of patients with scleroderma with autoantibodies against the minor spliceosome complex. The work described in this thesis will enhance our ability to study and exploit the properties of antibodies and the humoral immune response. / Biophysics

Primate lentiviruses (PLVs), including human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and the simian immunodeficiency viruses (SIVs), cause persistent lifelong infections despite the presence of virus-specific adaptive immune responses. The target of antibodies is the viral envelope glycoprotein (Env), which is expressed on the surface of virions and infected cells as a trimer of gp120:gp41 heterodimers. SIV env sequence variation arising from evasion of antibodies is well established and closely mimics the pattern observed in HIV-infected human patients, yet despite the experimental advantages of the macaque model, the viral dynamics during acute and early infection leading to escape within env have not been well defined. To examine the evolutionary dynamics of SIV env during early infection (< 6 months post-infection), we obtained longitudinal plasma samples from a small cohort of SIVmac251-infected rhesus macaques. We deep-sequenced the env gene from longitudinal samples spanning acute and early infection (2-29 weeks post-infection) from four SIV-infected rhesus macaques and the inoculum (a swarm stock of uncloned SIVmac251) that was used to infect the cohort (by repeated low-dose challenge). Using high-resolution next generation sequencing (NGS), we captured a population bottleneck at the point of transmission from the stock into each animal, tracked the subsequent emergence of Env diversity from the initially homogeneous population, and correlated changes with the onset of Env-specific antibodies. We identified a pattern of common substitutions, insertions, and deletions in env of animals with antibody responses, which repeatedly emerge in SIV-infected primate hosts, and found that adaptive changes clustered within short regions of the V1 and V4 loops of gp120. Surprisingly, we found multiple large in-frame deletions in V4 emerge to become dominant in the viral population in two animals with detectable antibody responses to Env. Furthermore, we developed a novel deep sequencing based viral fitness assay (Fit-Seq) and measured the relative fitness of several key in vivo antibody escape adaptations. Using Fit-Seq, we found that Env adaptations in V1 and V4 that confer antibody escape had no associated fitness costs in the absence of antibody, but rather, replicated to the same level as SIVmac239 WT. Surprisingly, we also found that Fit-Seq was able to detect antibody-mediated neutralization of SIVmac239 even in cases where activity was undetectable by standard neutralization assays. Our observation of reproducible patterns of Env variation clustered in V1 and V4, together with measurements of relative fitness, suggest that antibody responses can select for mutations that confer viral escape yet have little or no associated replicative fitness cost in the absence of antibody, even while providing a clear fitness advantage in the environment of constant antibody selection encountered in vivo. Thus, sites that can change with little or no impact on relative fitness may have evolved as an immune evasion mechanism to facilitate rapid and early escape from Env-specific antibody. / Medical Sciences