Virology Commons^™

Host Entry Factors For Human Coronaviruses, Taylor Heald-Sargent

Dissertations

Coronaviruses infect a diverse range of animals from birds to pigs and cats to humans. Coronaviruses employ RNA-based replicative processes and as such are genetically adaptable to acquire novel host ranges. A coronavirus from one species can jump to another by shifting its entry requirements. As we learned from the Severe Acute Respiratory Syndrome (SARS) pandemic, this shift in species can have detrimental consequences. Thus, it is imperative that we understand the virus-host interaction during the entry process.

This dissertation focuses on host entry factors that influence human coronavirus entry. Recently, a new class of proteases, the type II transmembrane …

Adenovirus Vectors As Potent Adjuvants In Vaccine Development, Kathleen Ann Mcguire

Dissertations

Due to their ability to activate the immune system, replication-defective Adenoviruses (Ad) are potential vaccine vectors for several pathogens. The proinflammatory response to Ad contributes to the response to vaccine antigens. We found that reactive oxygen species (ROS) are an important signal in the proinflammatory response to Ad. We identified that serotype 5 adenovirus (Ad5) elicits ROS by inducing mitochondrial membrane damage, a process that is dependent on endosomal membrane rupture and Cathepsin release. This mitochondrial dysfunction contributes to NLRP3 inflammasome- and NFκB-dependent innate immune activation. The ROS-dependent inflammatory response likely contributes to the adaptive immune response by supporting DC …

Coronavirus Proteases As Therapeutic Targets: Development Of Biosensors To Detect Inhibition Of Protease Activity And Separation Of The Multiple Functions Of Coronavirus Papain-Like Proteases, Andrew Kilianski

Dissertations

Coronaviruses are important human pathogens and have the potential to severely impact public health on an international scale. The emergence of SARS-CoV and MERS-CoV highlight the need for research to identify antivirals and vaccines against coronaviruses. To develop therapeutics against current and potentially emergent coronaviruses, I utilized two approaches targeting the proteases encoded within all coronaviruses. The papain-like protease and 3C-like protease of coronaviruses are responsible for cleaving viral polyproteins early during infection, and this step is required for viral replication. To quantitatively assess the inhibition by small-molecule compounds on MERS-CoV protease activity, I developed a luciferase-based biosensor to monitor …

Host Proteins Interact With The Hiv-1 Core To Facilitate And Restrict, Zana Lukic

Dissertations

Host cell proteins, termed restriction factors, which inhibit viral replication at various stages of the viral life cycle, determine the species-specific tropism of numerous retroviruses. Many members of the TRIM family of proteins act as viral restriction factors. One well-characterized example is the ability of TRIM5á from rhesus macaques (rhTRIM5á) to inhibit human immunodeficiency virus type-1 (HIV-1) soon after viral entry but prior to reverse transcription (RT). It is well established that the restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5á. Following the binding of the viral core, TRIM5á mediates an event or …

Multifunctional Coronavirus Papain-Like Proteases As Targets For Antiviral Therapeutics And Vaccines, Anna Maria Mielech

Dissertations

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle Ease Respiratory Syndrome coronavirus (MERS-CoV) pose a severe threat to humans because of high mortality. Despite the risk of coronavirus (CoV) emerging in the human population there are no antiviral drugs or vaccines to combat coronavirus infection. The focus of my dissertation was to study the multifunctionality of papain-like proteases (PLPs) encoded within coronavirus genomes to facilitate the development of antiviral drugs and vaccines. The viral PLPs are critical for processing the amino-terminal end of the replicase during virus replication and are attractive targets for antiviral therapies.

In my research, I analyzed …

Reconsidering The Model Of Trim5Α Assembly: The Role Of The Linker2 (L2) Region In Trim5Α Assembly, Laura Johnsen

Master's Theses

The TRIM5α protein from rhesus macaques (TRIM5αrh) exhibits a remarkable ability to potently inhibit infection by Human Immunodeficiency Virus Type-1 (HIV-1). Extensive studies have shown that TRIM5α is capable of self-associating at many levels, eventually leading to the formation of a hexameric assembly that can superimpose on the hexameric lattice of the HIV-1 capsid. The mechanism underlying the self-association of TRIM5α and the molecular determinants of self-association remain to be completely understood. In this study, we show that the Linker 2 (L2) region of TRIM5rh is important for dimerization and higher order self-association, both of which are independent processes. Additionally, …

Evading Innate And Adaptive Immunity During Adenovirus Cell Entry, Shauna Marvin

Dissertations

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and α_v integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs …

The Role Of Linker 2 (L2) Region In Rhtrim5Α Assembly And Hiv-1 Restriction, Jayalaxmi Sastri

Dissertations

The cellular restriction factor TRIM5alpha inhibits infection by numerous retroviruses in a species specific manner. TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) and a related protein TRIM-Cyp from Owl monkeys restrict infection by HIV-1 while human TRIM5α (huTRIM5alpha) restricts infection by N-tropic murine leukemia virus (N-MLV) but not HIV-1. Several models have been proposed for retroviral restriction by TRIM5 proteins (TRIM5alpha and TRIMCyp). These models collectively suggest that TRIM5 proteins mediate restriction by recognizing specific determinants in the viral capsid and directly binding the capsid. Following binding, the TRIM5 proteins self-associate into large assemblies around the viral capsid, which leads to …

Exosomes: Antiviral Agents In The Human Lung, Jennifer Alexandra Novak

Master's Theses

Hundreds of interferon effectors comprise the immediate host response to virus infection. One family of interferon stimulated genes is the IFITM (Interferon-Induced Transmembrane) proteins. IFITM3, in particular, has been shown to block virus cell entry of Influenza A Virus, SARS Coronavirus, and West Nile Virus, among others. IFITM3 has also been identified as an essential factor for host protection against Influenza A Virus in mice and humans. However, the mechanism by which IFITM3 functions remains unclear.

IFITM3 may function both intracellularly and extracellularly to block virus infection in the human lung. The extracellular milieu contains microscopic vesicles termed exosomes which …

Identifying And Characterizing The Degradative Pathway Of The Retroviral Restriction Factor Rhtrim5Α, Rachel Nelson

Master's Theses

Human immunodeficiency virus 1 (HIV-1) is a lentivirus that progresses to acquired immunodeficiency syndrome (AIDS). The protein TRIM5alpha from rhesus macaques (rhTRIM5alpha) restricts HIV-1 by blocking infection after entry of the virion into cells. Treatment of rhTRIM5alpha expressing cells with inhibitors to a cellular degradation pathway, the proteasome, partially relieves restriction but does not inhibit rhTRIM5alpha protein turnover. The role of a second degradation pathway, the autophagy-lysosomal pathway, in TRIM5alpha mediated restriction has not been explored.

In the present study, we demonstrate that rhTRIM5alpha is degraded by chaperone mediated autophagy (CMA). Inhibition of CMA alters rhTRIM5alpha localization and turnover, while …

Mechanisms Of Adenovirus Membrane Permeabilization, Oana Maier

Dissertations

For a successful infection to occur, a virus must first penetrate host cell membranes to access intracellular sites of viral replication. Currently the mechanism through which adenovirus, a non-enveloped, dsDNA virus, disrupts the endosomal membrane during cell entry is not well characterized.

Recent studies suggest that adenovirus protein VI, which is released from the interior of the capsid during cell entry, has all of the in vitro membrane lytic activity of the virion. We found that protein VI binds membranes via an amino-terminal 80 residue α-helical domain. Critical to this interaction are conserved hydrophobic and basic lysine residues within this …

Cofactors In Coronavirus Entry, Ana Shulla

Dissertations

Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells.

The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated …

Role Of Type Ii Transmembrane Serine Proteases In Coronavirus Production, Gitanjali Cuckemane Subramanya

Master's Theses

Proteolytic cleavage of Coronavirus spike proteins at the appropriate time and location results in efficient virus entry activation. Type II transmembrane serine proteases (TTSPs), specifically, transmembrane protease serine 2 (TMPRSS2) when expressed on target cells, enhance SARS coronavirus entry by activating spike cleavage. This study investigated the effect of TTSPs expressed in Coronavirus producer cells. Murine Hepatitis Virus strain A59 (MHV A59) viruses produced in the absence of TMPRSS2 required target cell protease activity - either cell surface serine proteases or endosomal acidophilic proteases - for entry. MHV A59 viruses produced in the presence of TMPRSS2 were less infectious overall, …

Coronavirus Replicase Proteins: Multifunctional Mediators Of Replication And Innate Immunity Evasion, Mark Anthony Clementz

Dissertations

Coronaviruses are positive-sense, single-stranded RNA viruses. The majority of the RNA encodes non-structural proteins (nsps) that are translated as a large polyprotein, which is cleaved by the papain-like (PLP) and picornavirus 3C-like (3CLpro) proteases. The nsps modify host membranes to produce double membrane vesicles (DMVs) upon which the replicase-transcriptase assembles and synthesizes viral RNA. nsp3, nsp4, and nsp6 are integral membrane proteins believed to be involved in DMV formation. Work presented here demonstrates that nsp4 is subjected to N-linked glycosylation and mutation of N258 to threonine in nsp4 confers a temperature sensitive phenotype to MHV-A59 infectious clone virus. This virus …

The Inhibitory Effects Of The Extracts Of Zingiber Plants On The Adsorption, Growth, And Replication Of Phage Lpp-1 In Cyanobacterium, Ebby Paul Jido

Master's Theses

No abstract provided.