Regulation of human immunodeficiency virus replication by 2',5'-oligoadenylate-dependent RNase L - PubMed (original) (raw)

Regulation of human immunodeficiency virus replication by 2',5'-oligoadenylate-dependent RNase L

R K Maitra et al. J Virol. 1998 Feb.

Abstract

Activation of RNase L by 2',5'-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.

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Figures

FIG. 1

FIG. 1

Construction of recombinant HIV containing RNase L cDNA in the sense and antisense orientations. The plasmids constructed or used for this study included NL4-3 (wild-type HIV-1 proviral DNA), NL4-3Δ_nef_ (272 bases deleted from nef), NL4-3/sRL (forward orientation RNase L cDNA cloned in place of a 272-bp segment of the nef gene), and NL4-3/aRL (reverse orientation RNase L cDNA cloned in place of a 272-bp segment of the nef gene).

FIG. 2

FIG. 2

Expression of RNase L cDNA from a recombinant HIV provirus suppresses HIV replication in transfected Jurkat cells (A) and PBL (B). The cells were transfected with pNL4-3 (○), pNL4-3/Δ_nef_ (◊), pNL4-3/aRL (▴), or pNL4-3/sRL (•). p24 antigen in the cell-free culture supernatant was collected at regular intervals and measured. d, days.

FIG. 3

FIG. 3

RNase L levels increase and then decline after transfection with NL4-3/sRL due to a deletion of RNase L coding sequence. (A) RNase L levels from Jurkat cells transfected with pNL4-3 (○), pNL4-3/Δ_nef_ (◊), pNL4-3/aRL (▴), or pNL4-3/sRL (•). The basal level of RNase L in untreated cells was an average of values obtained at 4, 16, and 24 days (d) of cell culture. (B) Upper panel, Western blot analysis of RNase L from transfected Jurkat cells probed with monoclonal antibody against human RNase L; lower panel, PCR amplification of the integrated RNase L cDNA. RL, 3 ng of RNase L; M, λ/_Hin_dIII molecular size markers (numbers to left are in kilobases). Plasmid names and times posttransfection in days are indicated.

FIG. 4

FIG. 4

Reduced levels of HIV mRNAs in Jurkat cells transfected with NL4-3/sRL. (A) Upper panel, Northern blot of HIV RNA from Jurkat cells at 48, 72, and 96 h posttransfection with pNL4-3/sRL, pNL4-3/Δ_nef_, or pNL4-3/aRL as indicated; lower panel, Northern blot of 18S rRNA. (B) Quantitation of total HIV mRNA with a PhosphorImager.

FIG. 5

FIG. 5

Overexpression of RNase L from NL4-3 recombinant proviral DNA (NL4-3/sRL) accelerates HIV-induced cell death. Jurkat cells were transfected with pNL4-3 (○), pNL4-3/Δ_nef_ (◊), pNL4-3/aRL (▴), or pNL4-3/sRL (•) or were mock transfected with salmon sperm DNA (□), and the percentage of apoptotic cells as a function of time was determined by in situ detection of DNA fragmentation. d, days.

FIG. 6

FIG. 6

Levels of RNase L in NL4-3 and NL4-3/aRL virus-infected Jurkat cells without or with interferon pretreatment (5,000 U per ml). Upper panel, quantitation of RNase L; lower panel, Western blot used to prepare graph shown in upper panel. d, days.

FIG. 7

FIG. 7

Replication of NL4-3 virus (○ and •) and pNL4-3/aRL virus (▵ and ▴) in the presence (• and ▴) or absence (○ and ▵) of 5,000 U of alpha interferon per ml in Jurkat cells (A) and PBL (B).

FIG. 8

FIG. 8

Control of HIV replication by RNase L expression plasmids in Jurkat cells. (A) Plasmid maps for pcDNAneo, pcDNAneo/sRL, and pcDNAneo/aRL; (B) plasmids cotransfected with NL4-3 in Jurkat cells. p24 antigen was measured as a function of time. NL4-3 proviral DNA cotransfected with salmon sperm DNA (○), pcDNAneo (▵), pcDNAneo/aRL (▴), or pcDNAneo/sRL (•). d, days; SV40, simian virus 40; RSV, Rous sarcoma virus.

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