In addition to the ability of DC to acquire computer virus in and support computer virus replication both and open reading frame as described . of MFI of EGFP fluorescence in HIV-1wt-EGFP reporter computer virus infected and EGFP plasmid transfected Jurkat T cells.(0.13 MB TIF) pone.0007470.s001.tif (128K) GUID:?10528D79-9124-4433-805D-B49A9ECCB3A3 Figure S2: DC infection vs antigen uptake. DC were cocultured with either HIV-1wt-EGFP reporter virus-infected Jurkat T cells or with Jurkat cells expressing EGFP protein. Post coculture, the cells were stained for DC-SIGN and analyzed by confocal microscopy. DC illness, signifies DC cells productively infected and expressing EGFP (was Nifenalol HCl measured by EGFP distribution throughout the cell); DC uptake, represents DC take up EGFP protein (show the punctate pattern). Red, shows DC-SIGN positive cells.(0.94 MB TIF) pone.0007470.s002.tif (922K) GUID:?43E93A11-B784-4D2C-96AD-364AD923E693 Abstract Dendritic cells (DC) play a pivotal part in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection capture virus particles via DC-SIGN and transfer the computer virus to the interacting na?ve T cells. This prompted us to request the query whether DC could acquire computer Nifenalol HCl virus from infected T cells during DC-T cell connection. To address this, we investigated the likely transfer of computer virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results show that DC acquire computer virus from infected T cells via antigen uptake Rabbit Polyclonal to RAB5C mechanism and this results in illness of DC with manifestation of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in illness of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data display that DC illness is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate computer virus. Intro HIV-1 infects macrophages, dendritic cells and T cells, which are also the key cells involved in inducing immune activation against invading pathogens , , . HIV-1 transmission, illness and dissemination are facilitated by both cell-free and cell-associated computer virus and infect T cells efficiently as Trojan Horses . In addition to the ability of DC to acquire computer virus in and support computer virus replication both and open reading framework as explained . The reporter computer virus derived from the plasmid offers allowed us to measure the manifestation and subcellular distribution of EGFP (driven by HIV-1 LTR) only in infected DC. Results offered here indicate the cell-associated computer virus was taken by DC and infected DC as early as 12 hours and was managed for more than six days, whereas cell free virus required 2C3 days to establish effective illness in DC. Illness of DC via infected T cell is dependent on T cell-DC contact and is self-employed of viral envelope and DC-SIGN. Furthermore, the percentage of DC illness is definitely directly correlated with the ability of DC to acquire cell-associated antigen, suggesting DC could acquire computer virus from the infected T cells through the antigen uptake process. Collectively, these studies for the first time indicate that HIV-1 taken up from the DC through the antigen uptake mechanisms establishes illness in DC. Results Illness of DC mediated by cell connected virus DC generated as explained in methods were cocultured with infected lymphocytes at a percentage of 291 (DC: uninfected PBL: infected PBL). Post coculture cells were stained for DC-SIGN, and EGFP+/DC-SIGN+ cells were determined by circulation cytometry. DC were gated based on part scatter and ahead scatter followed by doublet discrimination gating (Fig. 1A). Solitary cells that are double positive for DC-SIGN+ and EGFP+ were considered as productively infected DC (Fig. 1A). Results from coculture experiment show that 7.6% of DC were infected at 12 hours post coculture with infected lymphocytes, whereas cell free virus did not infect DC (0%) at this time point (Fig. 1A). Addition of cycloheximide (CHX) (10 g/ml) during coculture completely blocked illness of DC further confirming that EGFP manifestation in infected DC was due to synthesis, and not due to cell conjugates or cell fusion. Assessment of Mean Fluorescence Intensity (MFI) of EGFP in infected DC and infected lymphocytes present in the same coculture (Fig. 1B), shows that transcription of HIV-1 LTR driven in infected DC is significantly less compared to infected lymphocytes. DC illness was further confirmed by fluorescence microscopy Nifenalol HCl where, DC-SIGN positive cells were EGFP also positive (Fig. 1C) as recognized by the standard subcellular distribution of EGFP that is indicative of synthesized EGFP. To further validate illness in DC, integrated proviral DNA was measured in EGFP+ DC..