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
. 2013 Dec 17:10:157.
doi: 10.1186/1742-4690-10-157.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

Charline Giroud, Nathalie Chazal, Bernard Gay, Patrick Eldin, Sonia Brun, Laurence Briant  1
Affiliations

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

Charline Giroud et al. Retrovirology. .

Abstract

Background: Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle.

Results: NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions.

Conclusions: Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Infectivity of H89-NL4.3 and PKI-NL4.3. (A) Infectivity of normalized amounts of sucrose purified NL4.3, PKI-NL4.3 and H89-NL4.3 (10 ng p24 standardized to a final volume of 100 μL) was assayed in MAGIC-5B indicator cell lines. Control experiments consisted of mock infected cells (-) or NL4.3 infected cells maintained in the presence of 10 μM AZT. Infectivity was quantified by β-galactosidase activity in total cell lysate. Each value represents an average of three experiments performed in triplicate ± standard deviation. (B) Evaluation of viral samples contamination by co-purifying inhibitors. Supernatant (Sup.) of uninfected 293 cells maintained in medium alone (Mock) or cultured in the presence of H89 or Myr-PKI were subjected to gradient sucrose ultracentrifugation. Samples (100 μL) were then mixed with NL4.3 viruses (10 ng p24) produced from PKA proficient cells and the mixture was used to infect MAGIC-5B cells. Infectivity was measured as described in (A).
Figure 2
Figure 2
Phenotypes of HIV-1 lacking PKA activity in primary human cells. Infectivity of normalized amounts of sucrose purified NL4.3, PKI-NL4.3 and H89-NL4.3 (50 ng p24 standardized for 1x106 cells) was assayed in PBMCs, CD4+ lymphocytes. Similar experiments were performed using primary monocyte-derived macrophages as target cells and NL81.A, PKI-NL81.A and H89-NL81.A viruses. At day 6 post-infection, HIV-1 DNA levels were monitored by qPCR amplification. Control experiments consisted of mock infected cells (-) or NL4.3- or NL81.A-infected cells maintained in the presence of 10 μM AZT. Data represent the average of duplicates ± SD and are expressed as percentages of wild-type conditions.
Figure 3
Figure 3
Characterization of PKI-NL4.3 and H89-NL4.3 viral particles. (A) 293T cells expressing HIV-1 NL4.3 were maintained in the presence of medium alone or supplemented with Myr-PKI (PKI) or H89 inhibitors. Normalized amounts of cells lysates (left panel) or sucrose cushion purified viruses (right panel) were sequentially probed with rabbit anti-RT or anti-gp41 sera or anti-p24 mAbs. (B) Genomic RNA in viral particles was quantified by qRT-PCR. Values are expressed as percentages of NL4.3 values ± SD. (C) NL4.3, PKI-NL4.3 and H89-NL4.3 viral particles were imaged by electron microscopy. Bar = 100 nm. (D) The number of fully mature and aberrant viruses was counted in each sample and expressed as a percentage of total observation (NL4.3 n = 57; PKI-NL4.3 n = 105; H89-NL4.3 n = 18). Error bars represent 95% confidence intervals.
Figure 4
Figure 4
Quantification of genomic RNA and reverse transcription intermediates in cells infected with NL4.3, PKI-NL4.3 or H89-NL4.3 viruses. MAGIC-5B cells were infected with normalized amounts of NL4.3, PKI-NL4.3 or H89-NL4.3. Levels of genomic RNA (A) or (-)ssDNA or second strand transfer cDNA (B) in the infected cells were monitored by qRT-PCR or qPCR respectively. Control experiments consisted of mock infected cells (-) or NL4.3 infected cells maintained in the presence of 10 μM AZT. Values are expressed as percentage of NL4.3 conditions ± SD. Specificity of qRT-PCR detection of genomic RNA was controlled by preincubation of the cells for 30 min at 37°C in the presence of 1 μg/ml T20 fusion inhibitor before virus exposure.
Figure 5
Figure 5
Quantification of intrinsic RT activity contained in NL4.3, H89-NL4.3 and PKI-NL4.3 particles. (A) Normalized amounts of sucrose-purified NL4.3, PKI-NL4.3 or H89-NL4.3 viruses were lysed and subjected to RT reaction in the presence of exogenous poly(A)/oligo dT template/primer hybrid and dNTPs. (B) RT reactions were performed with lysate of NL4.3 viruses incubated in the presence of H89 (100 μM) or Myr-PKI (50 μM) inhibitors. Addition of nevirapine (Nev) to the reaction mixture was used as a negative control. RT activity was quantified by ELISA as described in the Material and Methods section. Results are the mean of three separate experiments performed in triplicate ± standard deviation.
Figure 6
Figure 6
Synthesis of (-)ssDNA in permeabilized NL4.3, H89-NL4.3 and PKI-NL4.3 viruses. (A) Sucrose purified NL4.3 viruses permeabilized with 0.1 mM Triton X-100 were subjected to ERT reactions run for 16 h in the presence or absence of dNTPs. Control experiments consisted of incubation of viruses with dNTPs and 20 μg/mL nevirapine (Nev). (-)ssDNA copy numbers were determined in each sample by qPCR. Values are expressed as absolute copy numbers. Error bars indicate standard deviations (n = 4). (B) PKI-NL4.3 or H89-NL4.3 particles were subjected to ERT reactions as described in (A). (C) and (D) Accessibility of ERT products synthesized in NL4.3, H89-NL4.3 and PKI-NL4.3 particles was monitored by addition 4 U DNase and 105 copies of a tracer plasmid to the reaction mixture. After DNase inactivation and nucleic acids extraction, (-)ssDNA and tracer plasmid copy numbers were determined by qPCR. Values are expressed as a percentage of DNA levels detected in no DNAse reactions.
Figure 7
Figure 7
Contribution of virus-associated PKA activity in endogenous reverse transcription. NL4.3 viruses were subjected to ERT experiments run in the presence of dNTPs and increasing concentrations of H89 (A) or Myr-PKI (B). For each reaction, nucleic acids were extracted and (-)ssDBNA production was quantified by qPCR amplification. DNA levels detected in ERT reactions performed in the presence of nevirapine are indicated by a dotted line. Data are mean values of duplicate experiments and are expressed as a percentage of WT conditions.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abbink TE, Berkhout B. HIV-1 reverse transcription initiation: a potential target for novel antivirals? Virus Res. 2008;10:4–18. - PubMed
    1. Mougel M, Houzet L, Darlix JL. When is it time for reverse transcription to start and go? Retrovirology. 2009;10:24. - PMC - PubMed
    1. Darlix JL, Godet J, Ivanyi-Nagy R, Fosse P, Mauffret O, Mely Y. Flexible nature and specific functions of the HIV-1 nucleocapsid protein. J Mol Biol. 2011;10:565–581. - PubMed
    1. Wu X, Liu H, Xiao H, Conway JA, Hehl E, Kalpana GV, Prasad V, Kappes JC. Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex. J Virol. 1999;10:2126–2135. - PMC - PubMed
    1. Gleenberg IO, Herschhorn A, Hizi A. Inhibition of the activities of reverse transcriptase and integrase of human immunodeficiency virus type-1 by peptides derived from the homologous viral protein R (Vpr) J Mol Biol. 2007;10:1230–1243. - PubMed

Publication types

Substances

LinkOut - more resources