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

Cheney KM, McKnight Á - PLoS ONE (2010)

Bottom Line: 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.Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect.Our results add to the understanding of HIV-1 restriction by IFNα.

View Article: PubMed Central - PubMed

Affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

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.

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Related in: MedlinePlus

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 [15]. 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.
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pone-0013521-g002: 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 [15]. 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.

Mentions: MDM cultures were treated with IFNα at various times prior to, at and post infection with HIV-1 strains 89.6 and BaL, previously shown to be highly sensitive to its inhibitory effects. The most potent restriction was observed when IFNα was present before the cells were infected (Fig. 2a). IFNα was still seen to have a modest effect on replication when added at the same time as the virus, but this soon decreased to low/negligible levels if IFNα was introduced after first round infection was complete (+24 hr). These results show that suitable expression levels of antiviral proteins or non-translated RNA can take at least 4 hr following induction by IFNα (up to 24 hr for maximum effect) and suggest that the resultant antiviral state is most effective at inhibiting the initial establishment of infection.


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

Cheney KM, McKnight Á - PLoS ONE (2010)

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 [15]. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958147&req=5

pone-0013521-g002: 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 [15]. 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.
Mentions: MDM cultures were treated with IFNα at various times prior to, at and post infection with HIV-1 strains 89.6 and BaL, previously shown to be highly sensitive to its inhibitory effects. The most potent restriction was observed when IFNα was present before the cells were infected (Fig. 2a). IFNα was still seen to have a modest effect on replication when added at the same time as the virus, but this soon decreased to low/negligible levels if IFNα was introduced after first round infection was complete (+24 hr). These results show that suitable expression levels of antiviral proteins or non-translated RNA can take at least 4 hr following induction by IFNα (up to 24 hr for maximum effect) and suggest that the resultant antiviral state is most effective at inhibiting the initial establishment of infection.

Bottom Line: 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.Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect.Our results add to the understanding of HIV-1 restriction by IFNα.

View Article: PubMed Central - PubMed

Affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus