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Identification and characterization ...

Gomila, Raul Carlos.

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Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses./
Author:	Gomila, Raul Carlos.
Description:	179 p.
Notes:	Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2353.
Contained By:	Dissertation Abstracts International67-05B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3217736
ISBN:	9780542692536

Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses.
Gomila, Raul Carlos.

Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses. - 179 p.

Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2353.

Thesis (Ph.D.)--Harvard University, 2006.

Dengue virus, a member of the Flaviviridae, is a single-stranded, positive-sense RNA virus. The 3'untranslated region (UTR) of dengue virus does not have a poly(A) tail, instead it has a terminal 3' structure of two stem loops (3'SL) This structure is well conserved among all flaviviruses, despite the absence of extensive primary nucleotide sequence homology. Several host factors have been identified as RNA binding proteins that interact with the flavivirus 3'SL. However, the functional significance of those interactions has not been elucidated. We undertook an independent search for host factors that interact with the dengue 3'SL and more importantly, aimed to define the functional significance of these interactions. We developed an RNA-affinity chromatography column for purifying interacting host proteins using in vitro transcribed dengue 3'SL coupled to a Sepharose 413 matrix. Incubation of the eluted proteins with the dengue 3'SL RNA produced several RNA-protein complexes in bandshift assays. The wild-type dengue 3'SL was an effective competitor in bandshift experiments, compared to an unrelated viral 3'UTR RNA. Specific mutations in the dengue 3'SL diminished its competitive activity, suggesting that the binding of the eluted proteins to the dengue 3'SL is specific. SDS-PAGE, followed by MALDI MS-TOF analysis identified several proteins enriched from the RNA column. Two of these proteins, RHA and NF90, belong to the family of double stranded RNA binding proteins, and were shown to bind the dengue 3' SL by northwestern blot. eEF1A, previously reported to bind the dengue 3' SL RNA, was not identified using the RNA affinity chromatography. RNAi-mediated depletion of RHA and NF90 protein levels (between 50-65% and 65-85%, respectively) was used to evaluate their possible functional role(s) in dengue virus replication. At these levels of protein depletion, the dengue NS3 protein levels were not statistically different from the control treated cells. Our results suggest that, using the experimental conditions described, depleting NF90 and RHA has no detectable effect on dengue virus replication. Alternate interpretations of the data include that the RNAi-mediated protein depletion was insufficient to elicit a phenotypic effect, or, that the depletion effect is detectable only at time points earlier than in our analysis.

ISBN: 9780542692536Subjects--Topical Terms:

1017734
Biology, Microbiology.

Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses.
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100 1 $a Gomila, Raul Carlos. $3 1917905
245 1 0 $a Identification and characterization of host factors that bind the conserved 3' terminal stem loop RNA of flaviviruses.
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500 $a Adviser: Lee Gehrke.
502 $a Thesis (Ph.D.)--Harvard University, 2006.
520 $a Dengue virus, a member of the Flaviviridae, is a single-stranded, positive-sense RNA virus. The 3'untranslated region (UTR) of dengue virus does not have a poly(A) tail, instead it has a terminal 3' structure of two stem loops (3'SL) This structure is well conserved among all flaviviruses, despite the absence of extensive primary nucleotide sequence homology. Several host factors have been identified as RNA binding proteins that interact with the flavivirus 3'SL. However, the functional significance of those interactions has not been elucidated. We undertook an independent search for host factors that interact with the dengue 3'SL and more importantly, aimed to define the functional significance of these interactions. We developed an RNA-affinity chromatography column for purifying interacting host proteins using in vitro transcribed dengue 3'SL coupled to a Sepharose 413 matrix. Incubation of the eluted proteins with the dengue 3'SL RNA produced several RNA-protein complexes in bandshift assays. The wild-type dengue 3'SL was an effective competitor in bandshift experiments, compared to an unrelated viral 3'UTR RNA. Specific mutations in the dengue 3'SL diminished its competitive activity, suggesting that the binding of the eluted proteins to the dengue 3'SL is specific. SDS-PAGE, followed by MALDI MS-TOF analysis identified several proteins enriched from the RNA column. Two of these proteins, RHA and NF90, belong to the family of double stranded RNA binding proteins, and were shown to bind the dengue 3' SL by northwestern blot. eEF1A, previously reported to bind the dengue 3' SL RNA, was not identified using the RNA affinity chromatography. RNAi-mediated depletion of RHA and NF90 protein levels (between 50-65% and 65-85%, respectively) was used to evaluate their possible functional role(s) in dengue virus replication. At these levels of protein depletion, the dengue NS3 protein levels were not statistically different from the control treated cells. Our results suggest that, using the experimental conditions described, depleting NF90 and RHA has no detectable effect on dengue virus replication. Alternate interpretations of the data include that the RNAi-mediated protein depletion was insufficient to elicit a phenotypic effect, or, that the depletion effect is detectable only at time points earlier than in our analysis.
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