Several approaches can be used for data analysis

Several approaches can be used for data analysis. or the need for expensive data analysis software. Beyond the described assay, the Overlap Intensity macro includes adjustable settings for use in other methods requiring quantification of overlapping fluorescent signals. strong class=”kwd-title” Keywords: HIV-1, capsid, uncoating, ImageJ, image quantification, fluorescent microscopy, fluorescent overlap Rabbit Polyclonal to FAKD2 1. Introduction The viral capsid is critical for multiple early steps of HIV-1 replication. The conical capsid is made of ~1500 monomers of the viral capsid (CA) protein which surrounds the genomic RNAs and other associated proteins to form the viral core [1,2]. The capsid is implicated in the protection of the reverse transcription complex (RTC), microtubule assisted transport, and nucleoporin interactions [3,4,5,6]. The disassembly or remodeling of the capsid, a step termed uncoating, is a requirement for replication with hyperstable and unstable capsid mutants associated with decreased infectivity [3,7]. Furthermore, the process of uncoating also has an interplay with reverse transcription and nuclear import of the viral genome [8,9,10,11]. The ability to study the capsid and uncoating has improved due to recent advancements in the field. There are multiple assays available to study uncoating including capsid core stability assays, the fate of the capsid assay, the CsA washout assay, and fluorescence microscopy-based NQDI 1 uncoating NQDI 1 assays [1,3,12,13,14,15,16,17,18,19,20,21,22,23,24,25]. Each of these NQDI 1 assays has different strengths and weaknesses, so often a combination of assays is used to study the factors that influence uncoating. Several uncoating assays have been developed utilizing fluorescence microscopy with capsid detection by antibody staining or through the use of fluorescent markers [8,15,16,17,19,20,21,22]. Collectively, fluorescence microscopy techniques allow the process of uncoating to be studied in infected cells with direct NQDI 1 visualization of the capsid and the process of uncoating relative to other cellular structures or proteins. However, these assays are dependent of optimal fluorescent labelling and staining of virus. Some of these fluorescent microscopy assays may also focus on a bulk population of virions, some of which may be uninfectious. Most recently, fluorescent microscopy-based uncoating assays have been developed that utilize live cell NQDI 1 imaging to track the progression of early steps of replication (uncoating, reverse transcription, cytoplasmic transit, nuclear import) in single virions and, in some assays, infection of the target cell [15,16,17,22,23,24,25]. Despite the importance of these experiments, the equipment needed to maintain live cell imaging can be limiting to research institutions. A more accessible alternative to live microscopy-based experiments is the in situ uncoating assay, which relies on fixed cell imaging over a time course [8,19]. The in situ uncoating assay is definitely a confocal microscopy-based experiment where dual-labelled HIV-1 is used to infect cells. The dual-labelled computer virus includes either GFP-tagged Vpr or integrase (IN) viral proteins [18,26,27]. The GFP-tagged proteins associate with the viral core and act as a marker for its location in the cytoplasm and nucleus of the cell. In addition, the computer virus maker cells are transfected having a S15-dTomato plasmid. S15-dTomato includes the N-terminal section of c-Src which embeds into the cell membrane [27,28]. As progeny virions bud from maker cells, the tagged sponsor membrane is integrated into the HIV viral membrane. Inclusion of the S15-dTomato into the viral membrane allows fusion to be tracked with the loss of the dTomato transmission. In the in situ uncoating assay, cells are infected with this dual-labelled computer virus and then fixed over a time program. Fixed cells undergo antibody staining for CA having a Cy5-conjugated secondary antibody. The Cy5 signal allows the extent of uncoating to be quantified by either the percentage.