Soft jamming of viral particles in nanopores.

Viruses have remarkable physical properties and complex interactions with their environment. However, their aggregation in confined spaces remains unexplored, although this phenomenon is of paramount importance for understanding viral infectivity. Using hydrodynamical driving and optical detection, we developed a method to detect the transport of single virus in real time through synthetic nanopores.

Murine leukemia virus (MLV) P50 protein induces cell transformation via transcriptional regulatory function.

Background: The murine leukemia virus (MLV) has been a powerful model of pathogenesis for the discovery of genes involved in cancer. Its splice donor (SD')-associated retroelement (SDARE) is important for infectivity and tumorigenesis, but the mechanism remains poorly characterized. Here, we show for the first time that P50 protein, which is produced from SDARE, acts as an accessory protein that transregulates transcription and induces cell transformation.

Viro-fluidics: Real-time analysis of virus production kinetics at the single-cell level.

Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a "viro-fluidic" method, which combines microfluidics and virology at single-cell and single-virus resolutions.

Optical Quantification by Nanopores of Viruses, Extracellular Vesicles, and Nanoparticles.

Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type.

Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy.

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles.

Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection.

HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS). It affects millions of people worldwide and the pandemic persists despite the implementation of highly active antiretroviral therapy. A wide spectrum of techniques has been implemented in order to diagnose and monitor AIDS progression over the years.

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions.

Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM.

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds.

Current Peptide and Protein Candidates Challenging HIV Therapy beyond the Vaccine Era.

Human immunodeficiency virus (HIV) is a causative agent of acquired immune deficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) can slow down the replication of HIV-1, leading to an improvement in the survival of HIV-1-infected patients. However, drug toxicities and poor drug administration has led to the emergence of a drug-resistant strain.

Sensing of HIV-1 Entry Triggers a Type I Interferon Response in Human Primary Macrophages.

Along with CD4 T lymphocytes, macrophages are a major cellular source of HIV-1 replication and a potential viral reservoir. Following entry and reverse transcription in macrophages, cloaking of the viral cDNA by the HIV-1 capsid limits its cytosolic detection, enabling efficient replication. However, whether incoming HIV-1 particles are sensed by macrophages prior to reverse transcription remains unclear.

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging.

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity.

Imaging HIV-1 RNA dimerization in cells by multicolor super-resolution and fluctuation microscopies.

Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches.

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions.

Insights into the nuclear export of murine leukemia virus intron-containing RNA.

The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses.

HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus.

HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions.

MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs.

Background: Eukaryotic cells have evolved stringent proofreading mechanisms to ensure that intron-containing mRNAs do not leave the nucleus. However, all retroviruses must bypass this checkpoint for replication. Indeed, their primary polycistronic transcript (Full-Length) must reach the cytoplasm to be either translated or packaged as genomic RNA in progeny viruses.

MoMuLV and HIV-1 nucleocapsid proteins have a common role in genomic RNA packaging but different in late reverse transcription.

Retroviral nucleocapsid proteins harbor nucleic acid chaperoning activities that mostly rely on the N-terminal basic residues and the CCHC zinc finger motif. Such chaperoning is essential for virus replication, notably for genomic RNA selection and packaging in virions, and for reverse transcription of genomic RNA into DNA. Recent data revealed that HIV-1 nucleocapsid restricts reverse transcription during virus assembly--a process called late reverse transcription--suggesting a regulation between RNA packaging and late reverse transcription.

A pyrophosphatase activity associated with purified HIV-1 particles.

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate.

Cell biology of retroviral RNA packaging.

Generation of infectious retroviral particles rely on the targeting of all structural components to the correct cellular sites at the correct time. Gag, the main structural protein, orchestrates the assembly process and the mechanisms that trigger its targeting to assembly sites are well described. Gag is also responsible for the packaging of the viral genome and the molecular details of the Gag/RNA interaction are well characterized.

In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.

RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA.

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes.

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers.

Implications of the nucleocapsid and the microenvironment in retroviral reverse transcription.

This mini-review summarizes the process of reverse-transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on reverse-transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of reverse-transcription and hence virus infectivity.

Uracil DNA Glycosylase 2 negatively regulates HIV-1 LTR transcription.

Numerous cellular factors belonging to the DNA repair machineries, including RAD18, RAD52, XPB and XPD, have been described to counteract human immunodeficiency virus type 1 (HIV-1) replication. Recently, Uracil DNA glycosylase 2 (UNG2), a major determinant of the uracil base excision repair pathway, was shown to undergo rapid proteasome-dependent degradation following HIV-1 infection. However, the specific role of intracellular UNG2 depletion during the course of HIV-1 infection is not clearly understood.

When is it time for reverse transcription to start and go?

Upon cell infection by a retrovirus, the viral DNA polymerase, called reverse transcriptase (RT), copies the genomic RNA to generate the proviral DNA flanked by two long terminal repeats (LTR). A discovery twenty years ago demonstrated that the structural viral nucleocapsid protein (NC) encoded by Gag is an essential cofactor of reverse transcription, chaperoning RT during viral DNA synthesis. However, it is only recently that NC was found to exert a control on the timing of reverse transcription, in a spatio-temporal manner.