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Abstract We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery.

Abstract Background Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells. Results Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells. Conclusion Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription. [ABSTRACT FROM AUTHOR]

We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery. [ABSTRACT FROM AUTHOR]

Background: Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells. Results: Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells. Conclusion: Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages. [ABSTRACT FROM AUTHOR]

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

RbpA is an RNA polymerase (RNAP)-binding protein whose presence increases the tolerance levels of Mycobacteria to the first-line anti-tuberculosis drug rifampicin by an unknown mechanism. Here, we show that the role of Mycobacterium tuberculosis RbpA in resistance is indirect because it does not affect the sensitivity of RNAP to rifampicin while it stimulates transcription controlled by the housekeeping σ(A)-factor. The transcription regulated by the stress-related σ(F) was not affected by RbpA. The binding site of RbpA maps to the RNAP β subunit Sandwich-Barrel Hybrid Motif, which has not previously been described as an activator target and does not overlap the rifampicin binding site. Our data suggest that RbpA modifies the structure of the core RNAP, increases its affinity for σ(A) and facilitates the assembly of the transcriptionally competent promoter complexes. We propose that RbpA is an essential partner which advantages σ(A) competitiveness for core RNAP binding with respect to the alternative σ factors. The RbpA-driven stimulation of the housekeeping gene expression may help Mycobacteria to tolerate high rifampicin levels and to adapt to the stress conditions during infection.

Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance because actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We discovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small-molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5(-/-) mice and found that they have increased trabecular bone mass with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop antiosteoporotic treatments., (Copyright © 2011 American Society for Bone and Mineral Research.)