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
. 2015 May;89(10):5350-61.
doi: 10.1128/JVI.00476-15. Epub 2015 Mar 4.

Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells

Amy E Hulme  1 , Z Kelley  1 , Deirdre Foley  1 , Thomas J Hope  2
Affiliations

Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells

Amy E Hulme et al. J Virol. 2015 May.

Abstract

During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2.

Importance: The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine the effect of two CA binding drugs (PF74 and BI2) on viral replication in infected cells. HIV-1 was susceptible to both drugs for hours after uncoating, suggesting that these drugs affect later steps of viral replication. High-resolution structured illumination microscopy (SIM) revealed that a subset of CA localized to viral complexes in the nuclei of cells. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating, which may facilitate later steps of viral replication and serve as a drug target.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Comparison of HIV uncoating and drug susceptibility. (A) To examine the relationship between drug susceptibility and uncoating, the CsA washout assay was performed in parallel with PF74 and BI2 drug addition assays in OMK cells. Nevirapine (NVP) addition was used as a drug control. Data were normalized to maximal infectivity for the CsA washout assay and to the no-drug control for the drug addition assays. Shown are results from a representative experiment, with error bars representing standard error of the mean. (B) Susceptibility of HIV to PF74 and BI2 over time was tested in HeLa P4R5 cells, which lack TRIM-CypA. Data were normalized to the no-drug control. Shown are results from an average of four experiments, with error bars representing standard error of the mean. (C) HIV susceptibility to PF74 and BI2 was tested up to 12 h in HeLa P4R5 cells. Data were normalized to the no-drug control. Shown are results from an average of three experiments, with error bars representing standard error of the mean.
FIG 2
FIG 2
Effect of PF74 and BI2 on HIV capsid integrity. VSV G-pseudotyped virus labeled with mCherry-VPR and a fluid-phase GFP marker was incubated with cells expressing HA-tagged TRIM5α in the presence of PF74, BI2, or the carrier control DMSO. mCherry-Vpr labeled virus localizing to TRIM bodies was counted and scored for GFP signal in 20 images from each condition. The percentage of viral complexes retaining GFP (A) and total number of viral complexes localizing to TRIM bodies (B) averaged from three independent experiments are presented, with error bars denoting standard error of the mean.
FIG 3
FIG 3
Effect of PF74 and BI2 on reverse transcription and infectivity. Real-time PCR was used to examine the effect of different concentrations of PF74 (A), BI2 (B), or nevirapine (C) on the process of reverse transcription in HELA P4R5 cells. An infectivity assay was also performed with the same drug concentrations. Data were normalized to the no-drug control then plotted on the same axis. Shown are results from a representative experiment from three independent experiments, with error bars representing standard error of the mean.
FIG 4
FIG 4
Uncoating kinetics of the CA mutant virus 5Mut. The uncoating of the wild type (HIV-GFP) and the CA mutant virus 5Mut was tested using the CsA washout assay. (A) 5Mut virus uncoats with kinetics similar to that of wild type. Shown are results from a representative assay, with error bars denoting standard error of the mean. (B) 5Mut virus has a half-life of uncoating similar to that of the wild type. Shown is the average half-life of uncoating, calculated from three independent experiments, with error bars denoting standard error of the mean.
FIG 5
FIG 5
Effect of CA mutations on drug susceptibility. The drug addition assay with PF74 (A and B), BI2 (C and D), or nevirapine (E and F) was performed over an extended time course with CA mutant viruses N74D, E45A, Q63/67A, A92E, and 5Mut. For each virus, data were normalized to the no-drug control. Shown are results from an average of three experiments, with error bars representing standard error of the mean.
FIG 6
FIG 6
Association of CA with viral complexes in cells. (A and B) High-resolution structural imaging was used to detect the presence of CA localizing to viral complexes in CHO cells. CHO cells were exposed to integrase-GFP-labeled virus (green), fixed, and stained for CA (red) and lamin to visualize the nuclear membrane (blue). Shown are examples of viral complexes localizing to the nucleus in a single z section (left), with a highlighted box presented as a volume projection in the z plane (right). The surface of the nucleus closest to the coverslip glass is labeled. (C to F) Control reactions for the high-resolution structural imaging experiment included an experimental sample exposed to bafilomycin A (C), integrase-GFP-labeled virus spun onto glass (D), an experimental sample stained only with secondary antibodies against human and rabbit (E), and a sample not infected with integrase-GFP-labeled virus (F). Shown is a single z section for each control. These images are the corresponding controls for the experiment presented in Fig. 7A and are displayed at the channel intensities used to analyze images for that experiment. (G and H). Deconvolution microscopy was used to detect the presence of CA localizing to viral complexes in HeLa cells. HeLa cells were exposed to integrase-GFP-labeled virus (green), fixed, and stained for CA (red) and lamin to visualize the nuclear membrane (blue). Shown is a single z section (left), with a highlighted box magnified to the right of the image.
FIG 7
FIG 7
Quantitation of CA signal associated with viral complexes. High-resolution structural imaging was used to detect the presence of CA localizing to viral complexes in CHO cells, and the intensity of the CA signal associated with each viral complex was determined. (A) Scatter plot of total CA intensity from a representative experiment, with each dot representing one viral complex counted. CA intensity was determined across a 4- by 4-pixel region for each viral complex. Localization of the viral complex was determined relative to the nuclear membrane. Baf control and virus on glass are virions randomly chosen in the bafilomycin A and virus-on-glass controls, respectively. The black bar is the median of each column. The dashed line is the level of background in the CA staining channel as assayed from the secondary-antibody control. (B) Scatter plot of total CA intensity from 3 independent experiments after normalization using the median CA intensity of the bafilomycin control for each experiment. CA intensity was determined across a 4- by 4-pixel region from each viral complex. Localization of the viral complex was determined relative to the nuclear membrane. Baf control are cytoplasmic virions randomly chosen in the bafilomycin A control. The black bar is the median of each column. The dashed line is the level of background in the CA staining channel as assayed from the secondary-antibody control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. von Schwedler UK, Stray KM, Garrus JE, Sundquist WI. 2003. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 77:5439–5450. doi:10.1128/JVI.77.9.5439-5450.2003. - DOI - PMC - PubMed
    1. Forshey BM, von Schwedler U, Sundquist WI, Aiken C. 2002. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol 76:5667–5677. doi:10.1128/JVI.76.11.5667-5677.2002. - DOI - PMC - PubMed
    1. Fitzon T, Leschonsky B, Bieler K, Paulus C, Schroder J, Wolf H, Wagner R. 2000. Proline residues in the HIV-1 NH2-terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication. Virology 268:294–307. doi:10.1006/viro.1999.0178. - DOI - PubMed
    1. Tang S, Murakami T, Agresta BE, Campbell S, Freed EO, Levin JG. 2001. Human immunodeficiency virus type 1 N-terminal capsid mutants that exhibit aberrant core morphology and are blocked in initiation of reverse transcription in infected cells. J Virol 75:9357–9366. doi:10.1128/JVI.75.19.9357-9366.2001. - DOI - PMC - PubMed
    1. Tang S, Murakami T, Cheng N, Steven AC, Freed EO, Levin JG. 2003. Human immunodeficiency virus type 1 N-terminal capsid mutants containing cores with abnormally high levels of capsid protein and virtually no reverse transcriptase. J Virol 77:12592–12602. doi:10.1128/JVI.77.23.12592-12602.2003. - DOI - PMC - PubMed

Publication types

LinkOut - more resources