Interferon-alpha mediates restriction of human immunodeficiency virus type-1 replication in primary human macrophages at an early stage of replication

Abstract

Type I interferons (IFNα and β) are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα.

Figure 1. IFNα potently inhibits HIV-1 replication in MDM but not PBMC/MDDC.

(a) MDM, PBMC and MDDC were treated with 500 IU/ml IFNα 24 hr prior to challenge with HIV-1 89.6. Culture supernatants were assayed after 4 days for levels of RT by ELISA (pg/ml). (b & c) MDM were treated with 500 IU/ml IFNα or β 24 hr prior to infection with a panel of replication competent HIV-1. Infected foci were counted after 4 days. Fold reduction is the ratio of FFU/ml of no IFN compared to IFNα treated cells. Error bars represent SD of one representative experiment.

Figure 2. IFNα inhibits the establishment of infection and acts via unknown cellular protein/s.

MDM were treated with 500 IU/ml IFNα 24 hr prior to infection with replication competent HIV-1 89.6 and BaL. Infected foci were counted after 4 days. (a) IFNα was added at various time points prior to, at or after infection. (b) AZT (final concentration 100 nM) was added to cultures 24 hr post infection to block second round infection. (c) HEK 293T cells were co-transfected with pcDNA3.1-HA-tetherin and either WT or Δvpu 89.6 molecular clones. Cells were lysed and the presence or absence of tetherin was confirmed by Western blot using an anti-HA Ab. Tetherin is a 30–36 kDa protein that migrates as several species by SDS-PAGE, as a result of post-translational modifications . The levels of p24 protein were monitored as a loading control. (d) Supernatants from transfected HEK 293T cells were serially diluted and used to infect NP2-CD4-CXCR4 cells. Virus titres were determined after 48 hr. (e) MDM were challenged with tetherin resistant (WT) and tetherin sensitive (Δvpu) HIV-1 molecular clones ± IFNα. Data is presented as mean ± SD.

Figure 3. IFNα inhibits HIV-1 at an early post entry stage in the virus life cycle.

(a) MDM were treated with 500 IU/ml IFNα 24 hr prior to challenge with HIV-1 BaL. Cells were collected 0 and 24 hr post infection and total RNA was extracted. RT-qPCR was used to detect late HIV-1 products and results were normalised to GAPDH cDNA and compared with uninfected controls. Error bars represent SD of two independent experiments. (b) HIV-1 89.6Δenv was pseudotyped with VSV-G, RD114 and Ampho MLV envs and used to challenge MDM. Infected foci were counted after 4 days and are presented as mean ± SD.

Figure 4. Post entry restriction affects the kinetics of late HIV-1 RT products and integration in MDM but not PBMC/MDDC.

(a) MDM and (b) PBMC and MDDC were treated with 500 IU/ml IFNα 24 hr prior to challenge with HIV-1 BaL. Cells were collected at various time points after infection and DNA was extracted. Early (MDM), late (MDM, PBMC & MDDC) and integrated (MDM) HIV-1 DNA was normalised to genomic GAPDH and copy number is presented per 10⁶ cells. Data is presented as mean ± SD and is from one representative experiment.