Among viability assays that depend on the conversion of substrate to chromogenic product by live cells, the MTT assay is still among one of the most versatile and popular assays. The MTT assay involves the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is then solubilized and the concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to ∼10⁶ cells per well. As with the alamarBlue assay, small changes in metabolic activity can generate large changes in MTT, allowing one to detect cell stress upon exposure to a toxic agent in the absence of direct cell death. The assay has been standardized for adherent or nonadherent cells grown in multiple wells. The protocol uses a standard 96-well plate. This can be scaled up, however, to suit a different plate format. Plate 500–10,000 cells per well in a 96-well plate. The assay has good linearity up to 10⁶ cells.

TRIM5 is an E3 ubiquitin ligase with known antiretroviral restriction factor activity, although the mechanisms involved are poorly understood. Luban and colleagues now demonstrate that TRIM5 activates innate immune signalling pathways and acts as a pattern recognition receptor specific for the retrovirus capsid lattice. TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice3,4 and several reports indicate that TRIM5 has a role in signal transduction5,6,7, but the precise mechanism of restriction is unknown8. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13–UEV1A (also known as UBE2N–UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13–UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13–UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Algorithms that account for and overcome the intrinsic pixel-dependent readout noise from scientific complementary metal-oxide semiconductor (sCMOS) cameras prevent localization artifacts and avoid substantial compromises in localization uncertainty in single-molecule imaging. When combined with multi-emitter fitting, sCMOS cameras allow video-rate single-molecule switching nanoscopy. Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

A common method for determining cytotoxicity is based on measuring the activity of cytoplasmic enzymes released by damaged cells. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is found in all cells. LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged, a key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage. LDH activity can be easily quantified by using the NADH produced during the conversion of lactate to pyruvate to reduce a second compound in a coupled reaction into a product with properties that are easily quantitated. This protocol measures the reduction of a yellow tetrazolium salt, INT, by NADH into a red, water-soluble formazan-class dye by absorbance at 492 nm. The amount of formazan is directly proportional to the amount of LDH in the culture, which is, in turn, directly proportional to the number of dead or damaged cells.

Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ₂ ], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P ₂ was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P ₂ to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P ₂ interactions for particle release from cells.