Targets of small interfering RNA restriction during human immunodeficiency virus type 1 replication.
Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5alpha did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5' of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3'-to-5' siRNA amplification and spreading. In contrast, HIV-1 RNA 3' of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively "naked" cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5alpha, or other host restriction factors.
Gao
Yong
Y
Division of Infectious Diseases, BRB 1034, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
Lobritz
Michael A
MA
Roth
Justin
J
Abreha
Measho
M
Nelson
Kenneth N
KN
Nankya
Immaculate
I
Moore-Dudley
Dawn M
DM
Abraha
Awet
A
Gerson
Stanton L
SL
Arts
Eric J
EJ
D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D034741:RNA, Small Interfering; D012367:RNA, Viral