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


OBJECTIVE
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.
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Amy E
AE
Hulme
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Z
Z
Kelley
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Deirdre
D
Foley
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA thope@northwestern.edu.
Thomas J
TJ
Hope
D019380:Anti-HIV Agents / Q000494:pharmacology; D002213:Capsid / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000502:physiology*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology; D057646:Virus Uncoating / Q000187:drug effects / Q000502:physiology*
D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D007211:Indoles; C000591221:PF-3450074; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D010649:Phenylalanine