Abstract
Short interfering RNAs (siRNAs) have been used to inhibit HIV-1 replication. The durable inhibition of HIV-1 replication by RNA interference has been impeded, however, by a high mutation rate when viral sequences are targeted and by cytotoxicity when cellular genes are knocked down. To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. We used this library to select a comprehensive population of Jurkat T-cell clones, each expressing a single discrete shRNA. The Jurkat clones were then infected with HIV-1. Clones that survived viral infection represent moieties silenced for a human mRNA needed for virus replication, but whose chronic knockdown did not cause cytotoxicity. Overall, 252 individual Jurkat mRNAs were identified. Twenty-two of these mRNAs were secondarily verified for their contributions to HIV-1 replication. Five mRNAs, NRF1, STXBP2, NCOA3, PRDM2, and EXOSC5, were studied for their effect on steps of the HIV-1 life cycle. We discuss the similarities and differences between our shRNA findings for HIV-1 using a spreading infection assay in human Jurkat T-cells and results from other investigators who used siRNA-based screenings in HeLa or 293T cells.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, N.I.H., Intramural
MeSH terms
-
Antigens, Neoplasm / genetics
-
Antigens, Surface / genetics
-
Cell Line
-
Cell Proliferation
-
DNA-Binding Proteins / genetics
-
Exoribonucleases / genetics
-
Exosome Multienzyme Ribonuclease Complex
-
Flow Cytometry
-
Gene Library
-
Genome, Human*
-
HIV Core Protein p24 / genetics
-
HIV Core Protein p24 / metabolism
-
HIV-1 / genetics*
-
HIV-1 / growth & development
-
HIV-1 / metabolism
-
HeLa Cells
-
Histone Acetyltransferases / genetics
-
Histone-Lysine N-Methyltransferase
-
Humans
-
Jurkat Cells
-
Munc18 Proteins / genetics
-
Nuclear Proteins / genetics
-
Nuclear Receptor Coactivator 3
-
Nuclear Respiratory Factor 1 / genetics
-
Oligonucleotide Array Sequence Analysis / methods
-
Proteins / genetics*
-
RNA, Messenger / genetics
-
RNA, Small Interfering / genetics*
-
RNA-Binding Proteins
-
Trans-Activators / genetics
-
Transcription Factors / genetics
-
Virus Replication / genetics*
Substances
-
Antigens, Neoplasm
-
Antigens, Surface
-
DNA-Binding Proteins
-
EXOSC5 protein, human
-
HIV Core Protein p24
-
Munc18 Proteins
-
NRF1 protein, human
-
Nuclear Proteins
-
Nuclear Respiratory Factor 1
-
Proteins
-
RNA, Messenger
-
RNA, Small Interfering
-
RNA-Binding Proteins
-
STXBP2 protein, human
-
Trans-Activators
-
Transcription Factors
-
Histone-Lysine N-Methyltransferase
-
PRDM2 protein, human
-
Histone Acetyltransferases
-
NCOA3 protein, human
-
Nuclear Receptor Coactivator 3
-
Exoribonucleases
-
Exosome Multienzyme Ribonuclease Complex