Viral envelope proteins and the HIV-1 accessory gene Vpu mediate selectivity of viral and host proteins in retroviral assembly

Title from PDF of title page (University of Missouri--Columbia, viewed on May 14, 2013).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Dissertation advisor: Dr. Marc JohnsonIncludes bibliographical references.Vita.Ph. D. University of Missouri-Columbia 2012."May 2012."Retroviruses are enveloped RNA viruses that assembly primarily at the plasma membrane of the host cell. During budding from the membrane, they acquire their own glycoproteins as well as a lipid bilayer derived from the cell. The assembly process is complex and involves protein:protein, protein:RNA, and, likely, protein:lipid interactions between viral components and the cell. These interactions appear to be specific and retroviruses are selective in acquisition of proteins. Compatibility during retrovirus assembly appears to be mediated by multiple factors: physical compatibility between glycoproteins and viral structural proteins, trafficking of proteins to appropriate locations, lipid interactions between Gag, Env and the plasma membrane, and microdomain association. In addition to mediating coalescence of appropriate factors, retroviruses appear equally equipped at excluding select host cell proteins and have evolved a number of genes to do so. Surprisingly, retrovirus assembly has remained enigmatic and there are no anti-retroviral drugs that target the assembly stage of HIV replication. Much of the knowledge we have garnered on retrovirus assembly has been through a process known as pseudotyping, where the core structural proteins will accept the glycoproteins of an unrelated virus. Here we present work outlining contributions of the envelope protein from murine leukemia virus to assembly with the lentiviral vector human immunodeficiency virus-1 (HIV-1). We subsequently observed an interesting phenotype, where an HIV-1 accessory gene known as Vpu restricts the envelope protein from gibbon ape leukemia virus (GaLV Env) from assembling with HIV-1. Further studies from our lab demonstrated that Vpu recognizes GaLV Env in a manner almost identical to CD4, the natural cellular target of Vpu, and that GaLV Env is essentially a CD4 analogue. Interestingly, we have found that Vpu restricts both target proteins in a manner that does not fit with the previously described Vpu-restriction model for CD4. Collectively, the GaLV Env model offers a new tool for more carefully investigating how the HIV-1 accessory gene Vpu downmodulates the host cell receptor CD4.Includes bibliographical reference

Women\u27s Fitness Week

This project addressed women’s wellness. Many women experience physical and mental health issues related to a lack of physical activity or psychological support. Women’s Fitness Week was offered to women of the University of Akron and the outside community, providing opportunities to learn more about and improve upon the various dimensions of wellness – emotional, environmental, financial, intellectual, occupational, physical, social/cultural, and spiritual. Events were held at the University’s Student Recreation and Wellness Center and encompassed topics such as nutrition, weight training, and self-defense. The goals of this project were to: increase knowledge levels of fitness, health, and wellness, increase confidence levels, increase comfort levels at the Student Recreation and Wellness Center specifically, and ultimately give females the ability to take initiative and ownership of their health and fitness lifestyles and goals

The Interferon-stimulated gene Ifi27l2a restricts West Nile virus infection and pathogenesis in a cell-type and region-specific manner

The mammalian host responds to viral infections by inducing expression of hundreds of interferon-stimulated genes (ISGs). While the functional significance of many ISGs has yet to be determined, their cell type and temporal nature of expression suggest unique activities against specific pathogens. Using a combination of ectopic expression and gene silencing approaches in cell culture, we previously identified Ifi27l2a as a candidate antiviral ISG within neuronal subsets of the central nervous system (CNS) that restricts infection by West Nile virus (WNV), an encephalitic flavivirus of global concern. To investigate the physiological relevance of Ifi27l2a in the context of viral infection, we generated Ifi27l2a(−/−) mice. Although adult mice lacking Ifi27l2a were more vulnerable to lethal WNV infection, the viral burden was greater only within the CNS, particularly in the brain stem, cerebellum, and spinal cord. Within neurons of the cerebellum and brain stem, in the context of WNV infection, a deficiency of Ifi27l2a was associated with less cell death, which likely contributed to sustained viral replication and higher titers in these regions. Infection studies in a primary cell culture revealed that Ifi27l2a(−/−) cerebellar granule cell neurons and macrophages but not cerebral cortical neurons, embryonic fibroblasts, or dendritic cells sustained higher levels of WNV infection than wild-type cells and that this difference was greater under conditions of beta interferon (IFN-β) pretreatment. Collectively, these findings suggest that Ifi27l2a has an antiviral phenotype in subsets of cells and that at least some ISGs have specific inhibitory functions in restricted tissues. IMPORTANCE The interferon-stimulated Ifi27l2a gene is expressed differentially within the central nervous system upon interferon stimulation or viral infection. Prior studies in cell culture suggested an antiviral role for Ifi27l2a during infection by West Nile virus (WNV). To characterize its antiviral activity in vivo, we generated mice with a targeted gene deletion of Ifi27l2a. Based on extensive virological analyses, we determined that Ifi27l2a protects mice from WNV-induced mortality by contributing to the control of infection of the hindbrain and spinal cord, possibly by regulating cell death of neurons. This antiviral activity was validated in granule cell neurons derived from the cerebellum and in macrophages but was not observed in other cell types. Collectively, these data suggest that Ifi27l2a contributes to innate immune restriction of WNV in a cell-type- and tissue-specific manner

The interferon-induced exonuclease ISG20 exerts antiviral activity through upregulation of type I interferon response proteins

The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20−/− mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3′–5′ exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20−/− mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20−/− mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle

Diverse viral glycoproteins as well as CD4 co-package into the same human immunodeficiency virus (HIV-1) particles

BACKGROUND: Retroviruses can acquire not only their own glycoproteins as they bud from the cellular membrane, but also some cellular and foreign viral glycoproteins. Many of these non-native glycoproteins are actively recruited to budding virions, particularly other viral glycoproteins. This observation suggests that there may be a conserved mechanism underlying the recruitment of glycoproteins into viruses. If a conserved mechanism is used, diverse glycoproteins should localize to a single budding retroviral particle. On the other hand, if viral glycoproteins have divergent mechanisms for recruitment, the different glycoproteins could segregate into different particles. RESULTS: To determine if co-packaging occurs among different glycoproteins, we designed an assay that combines virion antibody capture and a determination of infectivity based on a luciferase reporter. Virions were bound to a plate with an antibody against one glycoprotein, and then the infectivity was measured with cells that allow entry only with a second glycoprotein. We tested pairings of glycoproteins from HIV, murine leukemia virus (MLV), Rous sarcoma virus (RSV), vesicular stomatitis virus (VSV), and Ebola virus. The results showed that glycoproteins that were actively recruited into virions were co-packaged efficiently with each other. We also tested cellular proteins and found CD4 also had a similar correlation between active recruitment and efficient co-packaging, but other cellular proteins did not. CONCLUSION: Glycoproteins that are actively incorporated into HIV-1 virions are efficiently co-packaged into the same virus particles, suggesting that the same general mechanism for recruitment may act in many viruses

IRF-5-dependent signaling restricts Orthobunyavirus dissemination to the central nervous system

ABSTRACT Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3 , Irf5 , and Irf7 or in Irf5 alone. Deletion of Irf3 , Irf5 , and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5 −/− mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5 −/− mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro , since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. IMPORTANCE Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3 −/− Irf7 −/− (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar −/− mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV

Hawai‘i Forest Review: Synthesizing the Ecology, Evolution, and Conservation of a Model System

As the most remote archipelago in the world, the Hawaiian Islands are home to a highly endemic and disharmonic biota that has fascinated biologists for centuries. Forests are the dominant terrestrial biome in Hawai‘i, spanning complex, heterogeneous climates across substrates that vary tremendously in age, soil structure, and nutrient availability. Species richness is low in Hawaiian forests compared to other tropical forests, as a consequence of dispersal limitation from continents and adaptive radiations in only some lineages, and forests are dominated by the widespread Metrosideros species complex. Low species richness provides a relatively tractable model system for studies of community assembly, local adaptation, and species interactions. Moreover, Hawaiian forests provide insights into predicted patterns of evolution on islands, revealing that while some evidence supports “island syndromes,” there are exceptions to them all. For example, Hawaiian plants are not as a whole less defended against herbivores, less dispersible, more conservative in resource use, or more slow-growing than their continental relatives. Clearly, more work is needed to understand the drivers, sources, and constraints on phenotypic variation among Hawaiian species, including both widespread and rare species, and to understand the role of this variation for ecological and evolutionary processes, which will further contribute to conservation of this unique biota. Today, Hawaiian forests are among the most threatened globally. Resource management failures – the proliferation of non-native species in particular – have led to devastating declines in native taxa and resulted in dominance by novel species assemblages. Conservation and restoration of Hawaiian forests now rely on managing threats including climate change, ongoing species introductions, novel pathogens, lost mutualists, and altered ecosystem dynamics through the use of diverse tools and strategies grounded in basic ecological, evolutionary, and biocultural principles. The future of Hawaiian forests thus depends on the synthesis of ecological and evolutionary research, which will continue to inform future conservation and restoration practices

Immunoglobulin replacement products protect against SARS-CoV-2 infection in vivo despite poor neutralizing activity

Immunoglobulin (IG) replacement products are used routinely in patients with immune deficiency and other immune dysregulation disorders who have poor responses to vaccination and require passive immunity conferred by commercial antibody products. The binding, neutralizing, and protective activity of intravenously administered IG against SARS-CoV-2 emerging variants remains unknown. Here, we tested 198 different IG products manufactured from December 2019 to August 2022. We show that prepandemic IG had no appreciable cross-reactivity or neutralizing activity against SARS-CoV-2. Anti-spike antibody titers and neutralizing activity against SARS-CoV-2 WA1/2020 D614G increased gradually after the pandemic started and reached levels comparable to vaccinated healthy donors 18 months after the diagnosis of the first COVID-19 case in the United States in January 2020. The average time between production to infusion of IG products was 8 months, which resulted in poor neutralization of the variant strain circulating at the time of infusion. Despite limited neutralizing activity, IG prophylaxis with clinically relevant dosing protected susceptible K18-hACE2-transgenic mice against clinical disease, lung infection, and lung inflammation caused by the XBB.1.5 Omicron variant. Moreover, following IG prophylaxis, levels of XBB.1.5 infection in the lung were higher in FcγR-KO mice than in WT mice. Thus, IG replacement products with poor neutralizing activity against evolving SARS-CoV-2 variants likely confer protection to patients with immune deficiency disorders through Fc effector function mechanisms

The Clacton Spear: the last one hundred years

In 1911 an eminent amateur prehistorian pulled the broken end of a pointed wooden shaft from Palaeolithic-age sediments at a seaside town in Essex. This artefact, still the earliest worked wood to be discovered in the world, became known as the Clacton Spear. Over the past 100 years it has variously been interpreted as a projectile weapon, a stave, a digging stick, a snow probe, a lance, a game stake and a prod to ward off rival scavengers. These perspectives have followed academic fashions, as the popular views of early hominins have altered. Since discovery the Clacton spear has also been replicated twice, has undergone physical transformations due to preservation treatments, and has featured in two public exhibitions. Within this article the changing context of the spear, its parallels, and all previous conservation treatments and their impacts are assessed.© 2015 Royal Archaeological Institute. This is an Accepted Manuscript of an article published by Taylor & Francis in The Archaeological Journal on 3rd March 2015, available online: http://www.tandfonline.com/doi.org/10.1080/00665983.2015.1008839.The attached document is the author(’s’) final accepted/submitted version of the journal article. You are advised to consult the publisher’s version if you wish to cite from it

mRNA vaccine boosting enhances antibody responses against SARS-CoV-2 Omicron variant in individuals with antibody deficiency syndromes

Individuals with primary antibody deficiency (PAD) syndromes have poor humoral immune responses requiring immunoglobulin replacement therapy. We followed individuals with PAD after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination by evaluating their immunoglobulin replacement products and serum for anti-spike binding, Fcγ receptor (FcγR) binding, and neutralizing activities. The immunoglobulin replacement products tested have low anti-spike and receptor-binding domain (RBD) titers and neutralizing activity. In coronavirus disease 2019 (COVID-19)-naive individuals with PAD, anti-spike and RBD titers increase after mRNA vaccination but wane by 90 days. Those vaccinated after SARS-CoV-2 infection develop higher and more sustained responses comparable with healthy donors. Most vaccinated individuals with PAD have serum-neutralizing antibody titers above an estimated correlate of protection against ancestral SARS-CoV-2 and Delta virus but not against Omicron virus, although this is improved by boosting. Thus, some immunoglobulin replacement products likely have limited protective activity, and immunization and boosting of individuals with PAD with mRNA vaccines should confer at least short-term immunity against SARS-CoV-2 variants, including Omicron