Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion.

HIV-1 can infect T cells by cell-free virus or by direct virion transfer between cells through cell contact-induced structures called virological synapses (VS). During VS-mediated infection, virions accumulate within target cell endosomes. We show that after crossing the VS, the transferred virus undergoes both maturation and viral membrane fusion. Following VS transfer, viral membrane fusion occurs with delayed kinetics and transferred virions display reduced sensitivity to patient antisera compared to mature, cell-free virus. Furthermore, particle fusion requires that the transferred virions undergo proteolytic maturation within acceptor cell endosomes, which occurs over several hours. Rapid, live cell confocal microscopy demonstrated that viral fusion can occur in compartments that have moved away from the VS. Thus, HIV particle maturation activates viral fusion in target CD4+ T cell endosomes following transfer across the VS and may represent a pathway by which HIV evades antibody neutralization.

Visualizing cell-to-cell transfer of HIV using fluorescent clones of HIV and live confocal microscopy.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy.

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde-fixed, and methanol-fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations (e.g. 785, 1230, 1305, 1660 cm(-1)) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples to avoid potential misinterpretation of the data.

An adaptable, portable microarray reader for biodetection.

We have developed an inexpensive portable microarray reader that can be applied to standard microscope slide-based arrays and other array formats printed on chemically modified surfaces. Measuring only 19 cm in length, the imaging device is portable and may be applicable to both triage and clinical settings. For multiplexing and adaptability purposes, it can be modified to work with multiple excitation/emission wavelengths. Our device is shown to be comparable to a commercial laser scanner when detecting both streptavidin-biotin and antibody interactions. This paper presents the development and characterization of a handheld microarray imager and directly compares it with a commercial scanner.