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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 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.
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pone-0013521-g003: 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.

Mentions: The lack of p24 protein in the MDM detected by staining for FFU indicated that the IFNα mediated block of viral replication was in the early part of the life cycle and at least prior to protein translation. Furthermore to support this we isolated mRNA from MDM infected with HIV-1 BaL and performed qRT-PCR to determine whether the quantity of HIV-1 transcripts produced in the presence of IFNα was affected. HIV-1 mRNA levels were negligible in the presence of IFNα (Fig. 3a). These results support the observations in Fig. 1c that IFNα acts at an earlier stage in the viral life cycle.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013521-g003: 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.
Mentions: The lack of p24 protein in the MDM detected by staining for FFU indicated that the IFNα mediated block of viral replication was in the early part of the life cycle and at least prior to protein translation. Furthermore to support this we isolated mRNA from MDM infected with HIV-1 BaL and performed qRT-PCR to determine whether the quantity of HIV-1 transcripts produced in the presence of IFNα was affected. HIV-1 mRNA levels were negligible in the presence of IFNα (Fig. 3a). These results support the observations in Fig. 1c that IFNα acts at an earlier stage in the viral life cycle.

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