Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation
Authors
Ozen, AysegulFaculty Advisor
Celia A. Schiffer, Ph.D.Academic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Biochemistry and Molecular PharmacologyDocument Type
Doctoral DissertationPublication Date
2013-05-29Keywords
Dissertations, UMMSDrug Resistance, Viral
HIV Protease
HIV Protease Inhibitors
Hepacivirus
Viral Nonstructural Proteins
Viral Drug Resistance
HIV Protease
HIV Protease Inhibitors
Hepacivirus
Viral Nonstructural Proteins
Immunology and Infectious Disease
Molecular Biology
Structural Biology
Virology
Metadata
Show full item recordAbstract
Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.DOI
10.13028/M2JK5VPermanent Link to this Item
http://hdl.handle.net/20.500.14038/32031Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M2JK5V
Scopus Count
Collections
Related items
Showing items related by title, author, creator and subject.
-
Immunization with single-cycle SIV significantly reduces viral loads after an intravenous challenge with SIV(mac)239Jia, Bin; Ng, Sharon K; DeGottardi, M. Quinn; Piatak, Michael; Yuste, Eloisa; Carville, Angela; Mansfield, Keith G.; Li, Wenjun; Richardson, Barbra A.; Lifson, Jeffrey D.; et al. (2009-01-23)Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV(mac)239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4(+) T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV(mac)239. However, significantly lower viral loads and higher memory CD4(+) T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV(mac)239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.
-
A switch in translation mediated by an antisense RNARanade, Koustubh; Poteete, Anthony R. (1993-08-01)Antisense RNAs regulate expression of target genes in a variety of ways--transcription termination, translation initiation, and mRNA stability. We describe a case in which the target gene encodes two polypeptides, and antisense RNA causes a switch in its translation by selectively inhibiting synthesis of one of the polypeptides. Bacteriophage P22 is a temperate Salmonella phage; in the prophage state it expresses only a handful of its genes. One of these genes, sieB, aborts the lytic development of some phages. P22 itself is insensitive to the lethal effect of SieB because it harbors a determinant called esc. We show that the sieB gene encodes two polypeptides--SieB, which is the exclusion protein, and Esc, which is a truncated version of SieB that inhibits its action. Superinfecting P22 synthesizes an antisense RNA, sas, that inhibits synthesis of SieB but allows continued synthesis of Esc, thus allowing P22 to bypass SieB-mediated exclusion. This translational switch induced by sas RNA is essential to vegetatively developing P22; a mutation that prevents this switch causes P22 to commit SieB-mediated suicide. Finally, we show that P22's Esc allows it to circumvent the SieB-mediated exclusion system of bacteriophage lambda.
-
Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challengeAlpert, Michael D.; Rahmberg, Andrew R.; Neidermyer, William; Ng, Sharon K.; Carville, Angela; Camp, Jeremy V.; Wilson, Robert L.; Piatak, Michael Jr.; Mansfield, Keith G.; Li, Wenjun; et al. (2010-10-13)Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naive controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naive control animals.