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. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions.

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. RESULTS: By infecting cells with MLV particles containing SDARE transcript alone (lacking genomic RNA), we show that SDARE can spread to neighbouring cells as shown by the presence of P50 in infected cells. Furthermore, a role for P50 in cell transformation was demonstrated by CCK8, TUNEL and anchorage-independent growth assays. We identified the integrase domain of P50 as being responsible for transregulation of the MLV promoter using luciferase assay and RTqPCR with P50 deleted mutants. Transcriptomic analysis furthermore revealed that the expression of hundreds of cellular RNAs involved in cancerogenesis were deregulated in the presence of P50, suggesting that P50 induces carcinogenic processes via its transcriptional regulatory function. CONCLUSION: We propose a novel SDARE-mediated mode of propagation of the P50 accessory protein in surrounding cells. Moreover, due to its transforming properties, P50 expression could lead to a cellular and tissue microenvironment that is conducive to cancer development.

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. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 105 particles/mL for an acquisition time of 60 s with the ability to increase the acquisition time to achieve a lower limit.

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. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

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. Here, we show that HIV-1 triggers a broad expression of interferon (IFN)-stimulated genes (ISG) in monocyte-derived macrophages within a few hours after infection. This response does not require viral reverse transcription or the presence of HIV-1 RNA within particles, but viral fusion is essential. This response is elicited by viruses carrying different envelope proteins and thus different receptors to proceed for viral entry. Expression of ISG in response to viral entry requires TBK1 activity and type I IFNs signaling. Remarkably, the ISG response is transient but affects subsequent viral spread. Together, our results shed light on an early step of HIV-1 sensing by macrophages at the level of entry, which confers an early protection through type I IFN signaling and has potential implications in controlling the infection.IMPORTANCE HIV infection is restricted to T lymphocytes and macrophages. HIV-1-infected macrophages are found in many tissues of infected patients, even under antiretroviral therapy, and are considered a viral reservoir. How HIV-1 is detected and what type of responses are elicited upon sensing remain in great part elusive. The kinetics and localization of the production of cytokines such as interferons in response to HIV is of critical importance to understanding how the infection and the immune response are established. Our study provides evidence that macrophages can detect HIV-1 as soon as it enters the cell. Interestingly, this sensing is independent of the presence of viral nucleic acids within the particles but requires their fusion with the macrophages. This triggers a low interferon response, which activates an antiviral program protecting cells against further viral challenge and thus potentially limiting the spread of the infection.

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. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.

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. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism.

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. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA.

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.Murine leukemia virus (MLV) is a prototype of simple retroviruses with only two well-regulated splicing events that directly influence viral leukemogenic properties in mice. Several cis-elements have been identified in the FL RNA that regulate its cytoplasmic accumulation. However, their connection with an export mechanism is yet unknown. Our goal was to identify the cellular pathway used by MLV to export its RNAs into the cytoplasm of the host cells. RESULTS: Since other retroviruses use the CRM1 and/or the Tap/NXF1 pathways to export their unspliced RNA from the nucleus, we investigated the role of these two pathways in MLV replication by using specific inhibitors. The effects of export inhibition on MLV protein synthesis, RNA levels and RNA localization were studied by Western blotting, RT-qPCR, fluorescence microscopy and ribonucleoprotein immunoprecipitation assays. Taken together, our results show for the first time that MLV requires the Tap/NXF1-mediated export pathway, and not the CRM1 pathway, for the expression of its spliced and unspliced RNAs and for FL RNA nuclear export. CONCLUSIONS: By contrast to HIV-1, MLV recruits the same pathway for the cytoplasmic expression of its spliced and unspliced RNAs. Thus, MLV RNA expression depends upon coordinated splicing/export processes. In addition, FL RNA translation relies on Tap/NXF1-dependent export, raising the critical question of whether the pool of FL RNA to be packaged is also exported by Tap/NXF1.

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. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥104 VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.

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. Until recently, much less was understood about the cell biology of retrovirus RNA packaging. However, novel biochemical and live-cell microscopic approaches have identified where in the cell the initial events of genome recognition by Gag occur. These recent developments have shed light on the role played by the viral genome during virion assembly. Other central issues of the cell biology of RNA packaging, such as how the Gag-RNA complex traffics through the cytoplasm towards assembly sites, await characterization.

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. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.

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. Our study shows for the first time that overexpression of UNG2 inhibits HIV-1 replication. We demonstrate that this viral inhibition is correlated with a marked decrease in transcription efficiency as shown by monitoring HIV-1 LTR promoter activity and quantification of HIV-1 RNA levels. Interestingly, UNG2 inhibits LTR activity when stimulated by Tat transactivator or TNFalpha, while barely affected using Phorbol ester activation. Mutational analysis of UNG2 indicates that antiviral activity may require the integrity of the UNG2 catalytic domain. Altogether, our data indicate that UNG2 is likely to represent a new host defense factor specifically counteracted by HIV-1 Vpr. The molecular mechanisms involved in the UNG2 antiviral activity still remain elusive but may rely on the sequestration of specific cellular factor(s) critical for viral transcription.

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. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation.

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs.

Nucleocapsid mutations turn HIV-1 into a DNA-containing virus.

Retroviruses replicate by converting their positive sense genomic RNA into double-stranded DNA that is subsequently integrated into the host genome. This conversion is catalyzed by reverse transcriptase (RT) early after virus entry into the target cell and is chaperoned by the nucleocapsid protein (NC). In HIV-1, NC is composed of small basic domains flanking two highly conserved CCHC zinc fingers that specifically interact with the genomic RNA and RT. Through specific interactions with the genomic RNA and RT, and possibly with cellular factors, the NC zinc fingers were found to play critical roles in HIV-1 assembly and budding, and later in proviral DNA synthesis and integration. Therefore, intact NC zinc fingers are needed throughout the virus replication cycle. Here, we report for the first time that deleting either one or the two NC zinc fingers leads to an unexpected premature viral DNA synthesis in virus producer cells and the production of noninfectious particles with a high level of viral DNA. In addition to providing the first example of reverse transcription during the late steps of HIV-1 replication, these findings emphasize the fact that the NC zinc fingers are a major target for new drugs against HIV-1.

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. Besides the conventional approaches, microfluidics has provided useful methods for monitoring HIV-1 infection. In this review, we introduce continuous microfluidics as well as the fabrication and handling of microfluidic chips. We provide a review of the different applications of continuous microfluidics in AIDS diagnosis and progression and in the basic study of the HIV-1 life cycle.

Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles.

The viral genomes of alpha- and gamma-retroviruses follow an outbound route through the cytoplasm before assembling with the budding particle at the plasma membrane. We show here that murine leukemia virus (MLV) RNAs are transported on lysosomes and transferrin-positive endosomes. Transport on transferrin-positive vesicles requires both Gag and Env polyproteins. In the presence of Env, Gag is rerouted from lysosomes to transferrin-positive endosomes, and virion production becomes highly sensitive to drugs poisoning vesicular and endosomal traffic. Vesicular transport of the RNA does not require prior endocytosis, indicating that it is recruited directly from the cytosol. Viral prebudding complexes containing Env, Gag, and retroviral RNAs are thus formed on endosomes, and subsequently routed to the plasma membrane. This may allow retroviruses to hijack the endosomal machinery as part of their biosynthetic pathway. More generally, tethering to vesicles may provide an efficient mechanism for directed RNA transport.

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. This brief review summarizes findings on the timing of reverse transcription in wild type HIV-1 and in nucleopcapsid (NC) mutants where virions contain a large amount of newly made viral DNA. This brief review also proposes some explanations of how NC may control late reverse transcription during Gag assembly in virus producer cells.