Inhibition of human immunodeficiency virus type 1-mediated cytopathic effects by poly(L-lysine)-conjugated synthetic antisense oligodeoxyribonucleotides

Abstract

The ability of poly(L-lysine)-conjugated and methylphosphonate-modified synthetic human immunodeficiency virus type 1 (HIV-1) antisense oligodeoxyribonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection was studied. The abundance of viral antigens in oligomer-treated cultures indicated that the oligomers did not significantly affect viral infectivity. Similarly, no significant effects on relative viral RNA accumulation were apparent. The presence of poly(L-lysine)-modified oligomer complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus titre in infected cultures. In addition, these cells were protected from HIV-1-mediated cytopathic effects while the other cultures rapidly succumbed to the cytotoxic effects of HIV-1 infection. The presence of poly(L-lysine)-conjugated oligomer resulted in the establishment of a persistent HIV-1 infection characterized by a highly productive virus infection in the absence of cell death while treatment of persistently infected cells with phorbol ester resulted in renewed cytopathicity. These results demonstrate the ability of synthetic antisense oligonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection.

Fig 1.

Kinetics of viral replication in infected MT4 cells in the presence of antisense oligomers. MT4 cells were infected with HIV-1 and incubated with antisense oligomers as described in Methods. At the indicated times following infection, aliquots of culture supernatant and infected cells were removed for analysis of RT activity (a) and viral RNA levels by solution RNA hybridization (b) using single-stranded RNA probes generated from the LTR region of HIV-1 as shown in (c). +, Cells cultured without oligomer; ●, cells plus unmodified S.D. oligomer; ■, cells plus methylphosphonate-modified S.D. oligomer; ▲, cells plus poly(l-lysine)-modified S.D. oligomer.

Fig 2.

Inhibition of HIV-1 protein production in the presence of antisense oligomers. MT4 cells were infected and cultured with 1 μM parvovirus H-1 poly(l-lysine) oligomer (a); 200 nM-methylphosphonate-modified S.D. oligomer (b); and 200 nM-poly(l-lysine) S.D. oligomer (c). At 4 days p.i., cells were labelled for 3 h with [³⁵S]methionine and [³⁵S]cysteine and the viral proteins were immunoprecipitated with sera from a seronegative subject (lane 1) or sera from two HIV-1-seropositive asymptomatic haemophiliacs (lane 2,3) with titres of 5120 and 2560 respectively.

Fig. 3.

Southern blot analysis of HIV-1 DNA in persistently infected MT4 cells. Total cellular DNA was extracted from MT4 cells persistently infected with HIV-1, digested with BglII, HindIII, KpnI, PvuII, XbaI and EcoRI (lanes 1 to 6 respectively), blotted and hybridized with an HIV-1-specific genomic-length DNA probe.

Fig. 4.

Northern blot analysis of the CD4 mRNA levels in uninfected (lane 1) and persistently infected (lane 2) MT4 cells. Total cellular RNA (20 μg/lane) was denatured and electrophoresed through agarose gels containing formaldehyde and transferred to nylon membranes. The T4 probe was 3·0 kb cDNA pT4B (Maddon et al., 1986) containing the translated portion of the CD4 gene. β-Actin (Gunning et al., 1983) and β-tubulin (Hall et al., 1983) probes demonstrate equivalent amounts of RNA analysed in each lane.

Fig. 5.

TPA-mediated stimulation of HIV-1 replication and cytotoxicity in persistently infected MT4 cells. Persistently infected cells were incubated with 1 ng/ml TPA plus α-amanitin (1 μg/ml) for 24 h followed by analysis of viability (trypan blue exclusion, shaded), RT (unshaded) and viral RNA (hatched) levels at 24 h and 84 h post-TP A addition. Values are expressed relative to those obtained from cultures incubated without TPA.