Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr;20(4):425-9.
doi: 10.1038/nm.3489. Epub 2014 Mar 23.

New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo

C Korin Bullen  1 , Gregory M Laird  1 , Christine M Durand  2 , Janet D Siliciano  2 , Robert F Siliciano  3
Affiliations

New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo

C Korin Bullen et al. Nat Med. 2014 Apr.

Abstract

HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.

PubMed Disclaimer

Figures

Figure 1
Figure 1. LRAs do not induce outgrowth of latent HIV-1
(a) Schematic of LRA outgrowth assay. (b) LRA-treated rCD4s were stained with Annexin-V and 7-AAD. Toxicity was defined as percent positivity by flow cytometry. (c) Viral outgrowth from LRA-treated rCD4s from infected individuals. Wells positive by ELISA for HIV-1 p24 antigen at 14 days are depicted with a positive sign. Negative wells are depicted with a negative sign. (d) Culture supernatant HIV-1 mRNA (copies mL−1) from LRA-treated rCD4s obtained from five infected individuals (S26–S30). Dotted line indicates limit of detection (208.3 copies mL−1). Error bars indicate mean ± s.e.m.
Figure 2
Figure 2. LRAs do not consistently induce HIV-1 mRNA production in cells from HIV-1 infected individuals on ART
(a) Schematic of HIV-1 mRNA detection by RT-qPCR. Intracellular HIV-1 mRNA from LRA-treated rCD4s obtained from infected individuals presented as (b) fold change relative to DMSO control (mean ± s.e.m.) and (c) copies of HIV-1 mRNA per million rCD4 equivalents. Data points represent mean effect of the LRA for each individual. Statistical significance was determined using a paired t-test. RT(−) controls were negative for all samples. Lines connect data points from each infected individuals. Dotted line indicates limit of quantification (L.O.Q.) of 10 copies. Detectable values below L.O.Q. were assigned 10 copies. Undetectable values were assigned 1 copy.
Figure 3
Figure 3. A primary CD4+ T cell model of HIV-1 latency is responsive to LRAs
(a) Intracellular HIV-1 mRNA from LRA-treated BCL-2-transduced primary CD4+ T latency model cells. Changes are presented as fold induction relative to DMSO control (mean ± s.d.). (b) LRA-induced reactivation in latency models cells, defined as the percent GFP+ cells normalized to the effect of PMA/I treatment (mean ± s.d.) as measured by flow cytometry. Intracellular HIV-1 mRNA in serially diluted latency models cells, presented as (c) copies of HIV-1 mRNA per million rCD4 equivalents (mean ± s.d.) and (d) fold change relative to DMSO control (mean ± s.d.). An x indicates sample was below the limit of detection. RT(−) controls were negative for all samples. For a and b, statistical significance was determined using unpaired t-test. Asterisk indicates P value >0.05 (** indicates 10−2, *** indicates 10−3, **** indicates 10−4).
Figure 4
Figure 4. Vorinostat induces transcripts containing HIV-1 gag sequence but not HIV-1 mRNA in cells from HIV-1 infected individuals on ART
(a) Schematic of RT-qPCR detection of host/HIV-1 read-through transcripts (purple arrows), transcripts containing HIV-1 gag sequence (blue arrows), and HIV-1 mRNA (pink arrows). (b) Effect of vorinostat and PMA/I on intracellular HIV-1 read-through, gag-containing, and mRNA transcripts in rCD4s from five infected individuals, presented as fold change relative to DMSO control. (c) Schematic of Gag specific cDNA synthesis and qPCR detection of read-through transcripts. (d) Effect of vorinostat on read-through transcripts containing gag in from five infected individuals, presented as fold change relative to DMSO control. RT(−) controls were negative for all samples.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Finzi D, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278:1295–1300. - PubMed
    1. Wong JK, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science. 1997;278:1291–1295. - PubMed
    1. Chun TW, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA. 1997;94:13193–13197. - PMC - PubMed
    1. Siliciano JD, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med. 2003;9:727–728. - PubMed
    1. Strain MC, et al. Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence. Proc Natl Acad Sci USA. 2003;100:4819–4824. - PMC - PubMed

Publication types

MeSH terms