A CHARACTERIZATION OF KEY RESIDUES IN CLASS I VIRAL FUSION PROTEINS IMPORTANT FOR FUSOGENIC ACTIVITY

Viral fusion proteins are critical for viral entry and subsequent infection. Class I fusion proteins are characterized by synthesis as an inactive precursor requiring cleavage by a host cell protease to become fusion competent. Though vaccine and antiviral therapeutic developments often target the fusion protein, questions surrounding cleavage dynamics and protein stability remain. The work presented in this dissertation investigates specific regions of three class I viral fusion proteins in an effort to identify key residues involved in proteolytic processing and membrane fusion. The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) mediates receptor binding, facilitates fusion needed for viral entry, and drives cell–cell fusion. We demonstrate that S must be proteolytically processed at the S1/S2 border and within the S2 subunit in order to become fusion competent. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion. The transmembrane (TM) region of the HeV hendra virus fusion protein (F) has been shown to play a role in F protein stability and the overall trimeric association of F. Previous work classified β-branched residues within the C-terminal TM domain as important for F protein endocytosis, proteolytic processing, and protein stability. The work presented here completes the analysis of the HeV F TM and identifies specific residues that alter F protein function, suggesting a role for these residues in the fusion process. The respiratory syncytial virus (RSV) fusion protein (F) requires cleavage at two sites, separated by 27 amino acids. Cleavage at both sites results in a 27 amino acid fragment, termed Pep27. Previous work has provided conflicting results on the relative timing of when the two cleavage events occur. In addition, the fate of Pep27 is unclear. Examination of F cleavage kinetics in both infected and transfected systems over time determined that cleavage of both sites occurs within the secretory pathway as F is transported to the cell surface. We found that the deletion of Pep27 does not alter F function, but the mutation of N-linked glycosylation sites within Pep27 reduces both F surface expression and cell-cell fusion activity. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within the Pep27 play in the biological function of F. The work presented in this dissertation identifies residues within distinct regions of class I viral fusion proteins critical for fusion protein cleavage and stability, therefore impacting infection

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2-infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process

Direct Measurement of the Top Quark Mass at D0

We determine the top quark mass m_t using t-tbar pairs produced in the D0 detector by \sqrt{s} = 1.8 TeV p-pbar collisions in a 125 pb^-1 exposure at the Fermilab Tevatron. We make a two constraint fit to m_t in t-tbar -> b W^+bbar W^- final states with one W boson decaying to q-qbar and the other to e-nu or mu-nu. Likelihood fits to the data yield m_t(l+jets) = 173.3 +- 5.6 (stat) +- 5.5 (syst) GeV/c^2. When this result is combined with an analysis of events in which both W bosons decay into leptons, we obtain m_t = 172.1 +- 5.2 (stat) +- 4.9 (syst) GeV/c^2. An alternate analysis, using three constraint fits to fixed top quark masses, gives m_t(l+jets) = 176.0 +- 7.9 (stat) +- 4.8 (syst) GeV/C^2, consistent with the above result. Studies of kinematic distributions of the top quark candidates are also presented.Comment: 43 pages, 53 figures, 33 tables. RevTeX. Submitted to Phys. Rev.

Search for Charged Higgs Bosons in Decays of Top Quark Pairs

We present a search for charged Higgs bosons in decays of pair-produced top quarks using 109.2 +- 5.8 pb^-1 of data recorded from ppbar collisions at sqrt{s} = 1.8 TeV by the D0 detector during 1992-96 at the Fermilab Tevatron. No evidence is found for charged Higgs production, and most parts of the [m(H+),tan(beta)] parameter space where the decay t -> bH+ has a branching fraction close to or larger than that for t -> bW+ are excluded at 95% confidence level. Assuming m(t) = 175 GeV and sigma(ppbar -> ttbar) = 5.5 pb, for m(H+) = 60 GeV, we exclude tan(beta) 40.9.Comment: 11 pages, 3 figures, submitted to PR

Search for Electroweak Production of Single Top Quarks in ppbar Collisions

We present a search for electroweak production of single top quarks in the electron+jets and muon+jets decay channels. The measurements use ~90 pb^-1 of data from Run 1 of the Fermilab Tevatron collider, collected at 1.8 TeV with the DZero detector between 1992 and 1995. We use events that include a tagging muon, implying the presence of a b jet, to set an upper limit at the 95% confidence level on the cross section for the s-channel process ppbar->tb+X of 39 pb. The upper limit for the t-channel process ppbar->tqb+X is 58 pb.Comment: 11 pages, 2 figures. This is the published versio

Probing BFKL Dynamics in the Dijet Cross Section at Large Rapidity Intervals in ppbar Collisions at sqrt{s}=1800 and 630 GeV

Inclusive dijet production at large pseudorapidity intervals (delta_eta) between the two jets has been suggested as a regime for observing BFKL dynamics. We have measured the dijet cross section for large delta_eta in ppbar collisions at sqrt{s}=1800 and 630 GeV using the DO detector. The partonic cross section increases strongly with the size of delta_eta. The observed growth is even stronger than expected on the basis of BFKL resummation in the leading logarithmic approximation. The growth of the partonic cross section can be accommodated with an effective BFKL intercept of a_{BFKL}(20GeV)=1.65+/-0.07.Comment: Published in Physical Review Letter

Search for Squarks and Gluinos in Events Containing Jets and a Large Imbalance in Transverse Energy

Using data corresponding to an integrated luminosity of 79 pb-1, D0 has searched for events containing multiple jets and large missing transverse energy in pbar-p collisions at sqrt(s)=1.8 TeV at the Fermilab Tevatron collider. Observing no significant excess beyond what is expected from the standard model, we set limits on the masses of squarks and gluinos and on the model parameters m_0 and m_1/2, in the framework of the minimal low-energy supergravity models of supersymmetry. For tan(beta) = 2 and A_0 = 0, with mu < 0, we exclude all models with m_squark < 250 GeV/c^2. For models with equal squark and gluino masses, we exclude m < 260 GeV/c^2.Comment: 10 pages, 3 figures, Submitted to PRL, Fixed typo on page bottom of p. 6 (QCD multijet background is 35.4 events

Measurement of the WW Boson Mass

A measurement of the mass of the $W$ boson is presented based on a sample of 5982 $W \rightarrow e \nu$ decays observed in $p\overline{p}$ collisions at $\sqrt{s}$ = 1.8~TeV with the D\O\ detector during the 1992--1993 run. From a fit to the transverse mass spectrum, combined with measurements of the $Z$ boson mass, the $W$ boson mass is measured to be $M_W = 80.350 \pm 0.140 (stat.) \pm 0.165 (syst.) \pm 0.160 (scale) GeV/c^2$.Comment: 12 pages, LaTex, style Revtex, including 3 postscript figures (submitted to PRL

Search for a Fourth Generation Charge -1/3 Quark via Flavor Changing Neutral Current Decay

We report on a search for pair production of a fourth generation charge -1/3 quark (b') in pbar p collisions at sqrt(s) = 1.8 TeV at the Fermilab Tevatron using an integrated luminosity of 93 pb^-1. Both quarks are assumed to decay via flavor changing neutral currents (FCNC). The search uses the signatures gamma + 3 jets + mu-tag and 2 gamma + 2 jets. We see no significant excess of events over the expected background. We place an upper limit on the production cross section times branching fraction that is well below theoretical expectations for a b' quark decaying exclusively via FCNC for b' quark masses up to m(Z) + m(b).Comment: Eleven pages, two postscript figures, submitted to Physical Review Letter