A single-molecule view on retroviral replication,,

The human immunodeficiency virus (HIV) is a lentivirus that belongs to the family of Retroviridae, and causes the acquired immunodeficiency syndrome (AIDS). In 2009, the number of HIV-infected individuals, estimated at more than 33.3 million (www.unaids.org) which increases annually with two to three million annually. The current treatment of HIV infection is composed of a combination therapy of anti-retroviral agents which efficiently suppresses viral replication. However, due to the seemingly inevitable development of resistance to the existing antiviral cocktails, and the absence of an effective vaccine, the search for new drugs against new targets continues. As HIV has only a limited genomic it hijacks cellular proteins, referred to as co-factors, for the completion of the replication cycle. The laboratory of molecular virology and gene therapy is looking for new cellular cofactors of HIV integrase, the enzyme that integrates the viral cDNA stably into the genome of the infected cell. After reverse transcription of the viral RNA into cDNA in the cytoplasm of the host cell, IN associates the viral DNA with a number of other viral and cellular proteins to form the pre-integration complex (PIC), which is then transported through the nuclear membrane into the nucleus of the host cell. This transport is mediated by the cellular karyopherine transportin-SR2 (TRN-SR2). In the nucleus the PIC is tethered to the chromatin by the binding of the cellular protein LEDGF/p75 (lens epithelium-derived growth factor). Next, the viral DNA is stably integrated into the genome of the host by the catalytic activity of IN, after which new virus particles can be formed. Interactions between these viral proteins and cellular cofactors are attractive targets for the development of novel anti-retroviral agents. Both LEDGF/p75 and TRN-SR2 are at the moment studied by different research laboratories over the world. In 2010 a new class of antiviral molecules was published, the LEDGINs that disrupt the protein-protein interaction between LEDGF/p75 and HIV-1 IN and shows a strong antiviral activity in cell culture. Currently the LEDGINs are further developed for clinical use in collaboration with Pfizer. Despite is has been proven that TRN-SR2 plays a distinct role in the nuclear translocation step, a direct interaction of this protein with IN, has not yet been demonstrated in the context of the PIC in the infected cell. Since this is almost impossible to examine with standard molecular techniques, there is a need for a robust way to study protein-protein interactions of HIV on single-virus and / or single-PIC level in living cells during the early steps of the replication cycle of the virus. The expertise of the laboratory of photochemistry and spectroscopy is the development and application of quantitative fluorescence techniques, particularly techniques for single-molecule spectroscopy and microscopy. In this project, we want to develop and optimize a method to study the early steps of viral replication in a quantitative manner, at single-molecule level, in infected cells using fluorescence imaging. The intended techniques should enable us to characterize the stoichiometry and affinity of the protein-protein interactions between viral and cellular factors in time and space, which will provide crucial information about the interaction of viral proteins with co-factors such as TRN-SR2 and LEDGF / p75 in the search for new inhibitors of HIV replication.status: publishe

Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice

Imaging the Replication of Single Viruses: Lessons Learned from HIV and Future Challenges To Overcome.

The molecular composition of viral particles indicates that a single virion is capable of initiating an infection. However, the majority of viruses that come into contact with cells fails to infect them. Understanding what makes one viral particle more successful than others requires visualizing the infection process directly in living cells, one virion at a time. In this Perspective, we explain how single-virus imaging using fluorescence microscopy can provide answers to unsolved questions in virology. We discuss fluorescent labeling of virus particles, resolution at the subviral and molecular levels, tracking in living cells, and imaging of interactions between viral and host proteins. We end this Perspective with a set of remaining questions in understanding the life cycle of retroviruses and how imaging a single virus can help researchers address these questions. Although we use examples from the HIV field, these methods are of value for the study of other viruses as well.status: publishe

Molecular sieve activity of the nuclear pore during HIV-1 nuclear import revealed by single virus analysis

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Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Förster Resonance Energy Transfer Experiments

Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)­FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)­DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of single-labeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5–9 nm), we show that none of the dye pairs stands out: distance accuracies were generally <1 nm and precision was ∼0.5 nm. Finally, excitation of photoblinking dyes such as Alexa647 influences their fluorescence quantum yield, which has to be taken into account in distance measurements and leads to FRET dynamics. Although dye performance might differ in experiments on immobilized molecules, our combined ensemble and single-molecule approach is a robust characterization tool for all types of smFRET experiments. This is especially important when smFRET is used for atomic-scale distance measurements

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.status: publishe

Unraveling the dynamic interplay of HIV-1 integrase during its journey from the cytoplasm into the nucleus using fluorescence microscopy

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