Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

Günter Kraus, Rebeca Geffin, Gina Spruill, Andrea K. Young, Rachel Seivright, Diana Cardona, Jennifer Burzawa, H. James Hnatyszyn

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.

Original languageEnglish
Pages (from-to)3406-3411
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number6
DOIs
StatePublished - Mar 19 2002
Externally publishedYes

Fingerprint

Ribonuclease P
HIV-1
Messenger RNA
Guide RNA
Infection
HIV Core Protein p24
T-Lymphocytes
gag Gene Products
Giant Cells
Enzymes
Genes
Communicable Diseases
Plasmids
RNA
Gene Expression
Cell Line
Population
Neoplasms

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences. / Kraus, Günter; Geffin, Rebeca; Spruill, Gina; Young, Andrea K.; Seivright, Rachel; Cardona, Diana; Burzawa, Jennifer; Hnatyszyn, H. James.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 6, 19.03.2002, p. 3406-3411.

Research output: Contribution to journalArticle

Kraus, Günter ; Geffin, Rebeca ; Spruill, Gina ; Young, Andrea K. ; Seivright, Rachel ; Cardona, Diana ; Burzawa, Jennifer ; Hnatyszyn, H. James. / Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 6. pp. 3406-3411.
@article{51a372c5b8bb46b884d1b45991c9337a,
title = "Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences",
abstract = "RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.",
author = "G{\"u}nter Kraus and Rebeca Geffin and Gina Spruill and Young, {Andrea K.} and Rachel Seivright and Diana Cardona and Jennifer Burzawa and Hnatyszyn, {H. James}",
year = "2002",
month = "3",
day = "19",
doi = "10.1073/pnas.052651199",
language = "English",
volume = "99",
pages = "3406--3411",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "6",

}

TY - JOUR

T1 - Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

AU - Kraus, Günter

AU - Geffin, Rebeca

AU - Spruill, Gina

AU - Young, Andrea K.

AU - Seivright, Rachel

AU - Cardona, Diana

AU - Burzawa, Jennifer

AU - Hnatyszyn, H. James

PY - 2002/3/19

Y1 - 2002/3/19

N2 - RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.

AB - RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.

UR - http://www.scopus.com/inward/record.url?scp=0037133591&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037133591&partnerID=8YFLogxK

U2 - 10.1073/pnas.052651199

DO - 10.1073/pnas.052651199

M3 - Article

VL - 99

SP - 3406

EP - 3411

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 6

ER -