Katharine
Nina Bossart
Department of Microbiology and Immunology
Doctor of
Philosophy
2003
Major Advisor: Dr. Christopher C. Broder, Department of Microbiology and Immunology
Thesis Title: Structural and Functional Studies on the Fusion and Attachment Envelope Glycoproteins of Nipah Virus and Hendra Virus
ABSTRACT
Nipah virus (NiV) and Hendra (HeV) virus are emerging, biosafety level 4 paramyxoviruses responsible for fatal zoonotic infections of humans from pigs and horses, respectively, and are the prototypic members of a new Paramyxovirinae genus called Henipavirus. These enveloped, negative-sense RNA viruses infect cells through a pH-independent membrane fusion event mediated through the actions of their attachment (G) and fusion (F) envelope glycoproteins, which are also the principal antigens to which neutralizing antibodies are directed. Understanding the biological and functional features of the viral glycoproteins will help define the characteristics and properties of these novel viruses, and may provide insights into membrane fusion mechanisms, the virus infection process, and towards the development of therapeutics. Here, recombinant vaccinia virus vectors were generated to express the NiV and HeV glycoproteins. Glycoprotein functions and their cellular tropism characteristics were examined with a quantitative assay for membrane fusion. NiV and HeV glycoprotein-mediated fusion could be blocked by virus-specific antisera or synthetic peptides corresponding to the C-terminal a-helical heptad repeats of NiV or HeV F. Both F and G glycoproteins were required for membrane fusion and a broad species and cellular tropism pattern was observed for both HeV and NiV. Further, protease treatment of receptive host cells abolished viral glycoprotein-mediated fusion activity, suggesting a cell-surface protein serves as a receptor for these viruses. In addition, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but studies reported here show the NiV and HeV glycoproteins are capable of a highly efficient heterotypic functional activity amongst themselves, but not with other related paramyxoviruses. Finally, although closely related, these viruses were also distinguished from known paramyxoviruses by possessing a strong physical interaction between either viral F glycoprotein and the HeV G glycoprotein, while little interaction was noted between NiV G and either viral F. These later findings may provide a crucial new system for elucidating the paramyxovirus binding and entry process.
Donald
J. Chabot
Department of Microbiology and Immunology
Doctor of
Philosophy
2000
Major Advisor: Dr. Christopher Broder, Department of Microbiology and Immunology
Thesis Title: Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity
Abstract
CXCR4 and CCR5 are chemokine receptors and are coreceptors for human immunodeficiency virus (HIV-1). Host cells must express CD4 and a coreceptor for optimal HIV-1 entry. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex has been under intensive investigation. To define these regions we have employed an alanine-scanning mutagenesis strategy of the extracellular domains of CXCR4 coupled with a highly sensitive reporter-gene assay for HIV-l Env-mediated membrane fusion. Using a panel of 47 different CXCR4 mutations, we have identified several charged residues that appear important for coreceptor activity for X4 Envs: mutations El5A and E32A in the N-terminus, D97A in extracellular loop (ecl)-l, and Rl88A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. Mutation to alanine of one of the six tyrosines present in CXCR4, Y7, decreased coreceptor function. In addition, alanine substitution of any of the four extracellular cysteines alone resulted in conformational changes of varying degrees, while paired cysteine deletions could partially retain structure. Our data supports the notion that all four cysteines are involved in disulfide bond formation.
We have also identified substitutions that greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion: N11A, R30A, D187A, and D193A. Mutation of the aspartic acid at position 187 had the greatest effect on tropism. Mutation of an analogous serine in CCR5 to aspartic acid reduced CCR5 coreceptor activity with R5X4 and some R5 isolates. Mutation of the N11, a putative glycosylation site, had the second greatest effect on CXCR4 tropism. We determined that this site has a large carbohydrate moiety that is responsible for preventing CXCR4 from supporting R5 Env-mediated membrane fusion. Our data suggests the presence of conserved extracellular elements common to both CXCR4 and CCR5 involved in their coreceptor activities. These data will help to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains, and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.
Louise D.
Teel
Department of Microbiology and Immunology
Doctor of
Philosophy
2002
Major Advisor: Alison D. O'Brien, Ph.D., Department of Microbiology and Immunology
Title of Dissertation: The Regulation of Expression of the Stx2d Toxins in Shiga Toxin-producing Escherichia coli O91:H21 Strain B2F1
ABSTRACT
Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx2e from one human STEC isolate has been reported to be encoded within a toxin-converting phage. In this study, I examined O91:H21 STEC isolate B2F1 that carries two functional alleles (stx2d1 and stx2d2) for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages and other potential regulators of toxin expression. Mutants of B2F1 that produced one or the other Stx2d toxin were made. The Stx2d1-producing mutant (stx2d2::cat) was less cytotoxic for Vero cells than the Stx2d2-producing mutant (stx2d1::cat). Consistent with those results, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection, while the Stx2d2-producing mutant was nearly as virulent as wild-type B2F1. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. An stx2d1-containing lysogen was isolated from plaques on strain DH5a that had been exposed to lysates of the mutant that produced Stx2d1 only. However, that RecA- lysogen could not be induced for phage nor were culture lysates from it cytotoxic for Vero cells. By contrast, when the lysogen was transformed with a plasmid encoding RecA and induced with mitomycin C, culture extracts were cytotoxic for Vero cells. Furthermore, electron microscopic examination of extracts from the fB2F1-lysogen showed hexagonal particles that resembled the prototypic Stx2-converting phage 933W. These observations provide strong evidence that expression of Stx2d1 is bacteriophage-associated.
The
finding that synthesis of Stx2d1 but not Stx2d2 was associated with phage
induction led me to investigate regulation of Stx2d2 production. Transposon
mutagenesis of DH5a revealed genes associated with reduced expression of an
stx2d2 promoter::lacZ fusion in a reporter plasmid, observations that suggested
the inactivation of potential activators of transcription of stx2d2. The mutant
genes were isolated and sequenced. Two mutations identified were in caiD and
the "div" gene of DH5a; these mutants showed reduced cytotoxicity
and virulence for mice when transformed with an Stx2d2 toxin gene clone as
compared to wild-type DH5a similarly transformed. However, introduction of
the same mutations into strain B2F1 did not result in reduced cytotoxicity
or virulence. The influence of the caiD and"div" gene mutations
on toxin expression in DH5a may be polar and not correspond to the arrangement
of these genes in B2F1. Alternatively, the effects of these mutations may
only be evident when multiple copies of the toxin gene are present. In sum,
the regulation of expression of the two Stx2d alleles in B2F1 differs such
that Stx2d1expression is tightly repressed except under conditions that induce
the toxin-converting phage in which it is encoded. In contrast, Stx2d2 is
expressed at higher levels, independent of phage induction, under the direction
of some other yet-to-be-defined host factor. The DNA sequences that flank
stx2d2 share extensive homology to those flanking stx2d1 and other phage-borne
Stx genes, a finding that suggests that stx2d1 and stx2d2 share a common phage
origin but the phage sequences associated with stx2d2 are defective.