Of the HIV-1 capsid, uncoating remains poorly understood and is not
Of the HIV-1 capsid, uncoating remains poorly understood and is not currently possible to study by live-cell imaging techniques due to lack of available methods to label CA molecules without perturbing the function of the viral capsid. Fluorescence microscopic methods to track the core and viral protein or DNA components of the RTC/PIC have been employed previously [1,2,14-16], but are limited in terms of sensitivity. To circumvent these problems, we applied an alternative method of labeling HIV-1 RNA such that it could be stained with a fluorescent small molecule dye after capsid dissociation in vitro or during cell infection. The dye is specific for virus particle-associated RNA and can only access the viral nucleic acid after an initial uncoating step that appears to involve opening of the capsid. We observed that the kinetics of RNA staining were altered by the presence of rhTRIM5 or TRIMCyp, by CA substitutions altering capsid stability, and by addition of a small molecule inducer of HIV-1 uncoating. Surprisingly, the previously identified E45A CA mutant, which exhibits hyperstable cores in vitro, exhibited efficient staining of viral-associated RNA through cores in vitro and accelerated staining kinetics in vivo, suggestive of a permeable capsid.modified virus-associated RNA could, in principle, be both cellular and viral in origin. HIV-1 particles were made by transfecting 293T cells with a proviral construct and a plasmid encoding VSV-G in the presence of EU for different lengths of time to optimize vRNA labeling and normal viral protein production. EU was added to the transfection medium for the first 16 h of transfection, the last 24 h of transfection, or the entire 48 h of transfection. To test whether EU-labeled viruses were infectious, viruses produced in the presence of EU incorporation for different lengths of time were assayed for infectivity in HIV-1 indicator cells as well as for total CA production. For HIV-1 produced in the presence of 1 mM EU for 16 h, the specific infectivity, or infectious units (IU) per ng of CA antigen, was comparable to that of control virus made in the absence of EU (Table 1). Viruses produced in the presence of EU during the entire 48 h of transfection or for the last 24 h of transfection exhibited 2- and 3-fold lower infectivity relative to unlabeled HIV-1. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 Furthermore, decreased yields of virus were produced during extended culture time in EU, which was likely PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26162776 due to increased cellular toxicity of 293T cells incubated for longer periods of time with 1 mM EU (Additional file 1: Figure S1). Subsequent experiments were performed using 0.1 – 0.4 mM EU during 48 h of virus production to minimize cell toxicity. Viral DNA from cells infected with EU-labeled virus was sequenced and revealed no difference in mutations compared to unlabeled virus (data not shown), suggesting that EU incorporation does not affect reverse transcription. To determine if the EU-binding dye could penetrate intact cores, we treated EU-labeled HIV-1 particles with cell extraction buffer or buffer HIV-1 integrase inhibitor 2 dose containing lysates from 293T cells or 293T cells expressing rhTRIM5. The restriction factor rhTRIM5 has been reported to disrupt HIV-1 cores and assembled CA tubes in vitro [21-23]. To visualize stained and unstained particles, infectious virus containing a genome with MS2 protein binding sites was produced in cells expressing the MS2-GFP fusion protein and incubated with EU. Co-localization of vRNA staining and MS2-GFP was greate.