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Probing the mechanisms of fusion and...

Earp, Laurie Jean.

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Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A./
Author:	Earp, Laurie Jean.
Description:	184 p.
Notes:	Source: Dissertation Abstracts International, Volume: 64-03, Section: B, page: 1076.
Contained By:	Dissertation Abstracts International64-03B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3083079

Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A.
Earp, Laurie Jean.

Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A. - 184 p.

Source: Dissertation Abstracts International, Volume: 64-03, Section: B, page: 1076.

Thesis (Ph.D.)--University of Virginia, 2003.

Previous evidence (Hernandez et. al., 1997; Damico et. al, 1998) indicated that avian sarcoma/leukosis virus (ASLV) EnvA is activated for fusion by soluble receptor (sTva47) at neutral pH. These results were based upon studies using a soluble form of the EnvA glycoprotein (EnvA-PI) in an <italic>in vitro</italic> target membrane (liposome) binding assay. More recent evidence has suggested that there are two steps required to activate ASLV Envs for fusion: receptor binding at neutral pH, followed by exposure to low pH (Mothes et. al., 2000). This was based on the observed inhibition of ASLV DNA synthesis by lysosomotropic agents in a PCR-based assay, and the requirement of low pH for cell-cell fusion between Env- and receptor-expressing cells. In this dissertation, I describe experiments designed to directly test a requirement for low pH at different stages of ASLV-A fusion and entry. My results provide strong support that receptor binding at neutral pH is sufficient to activate ASLV EnvA such that the virus reaches the lipid mixing stage of fusion. More specifically, low pH is not required for target membrane binding of EnvA or ASLV-A particles, or for the exchange of lipids between pyrene-labeled ASLV-A particles and DF-1 cell membranes. Low pH does augment cell-cell fusion with receptor-expressing cells (Earp et. al., 2003). Furthermore, using a real-time (quantitative) PCR assay, I have confirmed that lysosomotropic agents (i.e., bafilomycin and NH<sub>4</sub>Cl) inhibit ASLV-A and ASLV-B DNA synthesis at low concentrations. Hence, a step between virus-cell membrane merger and production of reverse transcripts appears to require low pH. Future studies will address whether the low pH requirement is for a late stage of ASLV fusion (i.e., opening and expansion of a fusion pore) or for an early post-fusion event (i.e., uncoating).Subjects--Topical Terms:

1017734
Biology, Microbiology.

Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A.
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100 1 $a Earp, Laurie Jean. $3 1943012
245 1 0 $a Probing the mechanisms of fusion and cellular entry by a model retrovirus, ASLV-A.
300 $a 184 p.
500 $a Source: Dissertation Abstracts International, Volume: 64-03, Section: B, page: 1076.
500 $a Adviser: Judith White.
502 $a Thesis (Ph.D.)--University of Virginia, 2003.
520 $a Previous evidence (Hernandez et. al., 1997; Damico et. al, 1998) indicated that avian sarcoma/leukosis virus (ASLV) EnvA is activated for fusion by soluble receptor (sTva47) at neutral pH. These results were based upon studies using a soluble form of the EnvA glycoprotein (EnvA-PI) in an <italic>in vitro</italic> target membrane (liposome) binding assay. More recent evidence has suggested that there are two steps required to activate ASLV Envs for fusion: receptor binding at neutral pH, followed by exposure to low pH (Mothes et. al., 2000). This was based on the observed inhibition of ASLV DNA synthesis by lysosomotropic agents in a PCR-based assay, and the requirement of low pH for cell-cell fusion between Env- and receptor-expressing cells. In this dissertation, I describe experiments designed to directly test a requirement for low pH at different stages of ASLV-A fusion and entry. My results provide strong support that receptor binding at neutral pH is sufficient to activate ASLV EnvA such that the virus reaches the lipid mixing stage of fusion. More specifically, low pH is not required for target membrane binding of EnvA or ASLV-A particles, or for the exchange of lipids between pyrene-labeled ASLV-A particles and DF-1 cell membranes. Low pH does augment cell-cell fusion with receptor-expressing cells (Earp et. al., 2003). Furthermore, using a real-time (quantitative) PCR assay, I have confirmed that lysosomotropic agents (i.e., bafilomycin and NH<sub>4</sub>Cl) inhibit ASLV-A and ASLV-B DNA synthesis at low concentrations. Hence, a step between virus-cell membrane merger and production of reverse transcripts appears to require low pH. Future studies will address whether the low pH requirement is for a late stage of ASLV fusion (i.e., opening and expansion of a fusion pore) or for an early post-fusion event (i.e., uncoating).
590 $a School code: 0246.
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773 0 $t Dissertation Abstracts International $g 64-03B.
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791 $a Ph.D.
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856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3083079