In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

Palmitoylated Cysteines in Chikungunya Virus nsP1 Are Critical for Targeting to Cholesterol-Rich Plasma Membrane Microdomains with Functional Consequences for Viral Genome Replication.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

Neurocognitive impacts of arbovirus infections.

Arthropod-borne viruses or arbovirus, are most commonly associated with acute infections, resulting on various symptoms ranging from mild fever to more severe disorders such as hemorrhagic fever. Moreover, some arboviral infections can be associated with important neuroinflammation that can trigger neurological disorders including encephalitis, paralysis, ophthalmological impairments, or developmental defects, which in some cases, can lead to long-term defects of the central nervous system (CNS). This is well illustrated in Zika virus-associated congenital brain malformations but also in West Nile virus-induced synaptic dysfunctions that can last well beyond infection and lead to cognitive deficits. Here, we summarize clinical and mechanistic data reporting on cognitive disturbances triggered by arboviral infections, which may highlight growing public health issues spanning the five continents.

The Host DHX9 DExH-Box Helicase Is Recruited to Chikungunya Virus Replication Complexes for Optimal Genomic RNA Translation.

Beyond their role in cellular RNA metabolism, DExD/H-box RNA helicases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervariable domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV replication in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication.IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies.

Impact of HIV-1 Vpr manipulation of the DNA repair enzyme UNG2 on B lymphocyte class switch recombination.

BACKGROUND: HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modulation of several host cell functions. In addition to pro-apoptotic and cytostatic properties, Vpr can redirect cellular E3 ubiquitin ligases (such as DCAF1-Cul4A E3 ligase complex) to target many host proteins and interfere with their functions. Among them, Vpr binds the uracil DNA glycosylase UNG2, which controls genome uracilation, and induces its specific degradation leading to loss of uracil removal activity in infected cells. Considering the essential role of UNG2 in antibody diversification in B-cells, we evaluated the impact of Vpr on UNG2 fate in B lymphocytes and examined the functional consequences of UNG2 modulations on class switch recombination (CSR). METHODS: The impact of Vpr-induced UNG2 deregulation on CSR proficiency was evaluated by using virus-like particles able to deliver Vpr protein to target cells including the murine model CSR B cell line CH12F3 and mouse primary B-cells. Co-culture experiments were used to re-examine the ability of Vpr to be released by HIV-1 infected cells and to effectively accumulate in bystander B-cells. Vpr-mediated UNG2 modulations were monitored by following UNG2 protein abundance and uracil removal enzymatic activity. RESULTS: In this study we report the ability of Vpr to reduce immunoglobulin class switch recombination (CSR) in immortalized and primary mouse B-cells through the degradation of UNG2. We also emphasize that Vpr is released by producing cells and penetrates bystander B lymphocytes. CONCLUSIONS: This work therefore opens up new perspectives to study alterations of the B-cell response by using Vpr as a specific CSR blocking tool. Moreover, our results raise the question of whether extracellular HIV-1 Vpr detected in some patients may manipulate the antibody diversification process that engineers an adapted response against pathogenic intruders and thereby contribute to the intrinsic B-cell humoral defect reported in infected patients.

[Architecture and biogenesis of positive-stranded RNA virus replication organelles]. / Architecture et biogenèse des organelles de réplication des virus à ARN de polarité positive.

The replication of viral pathogens relies on their ability to manipulate their host. Several steps of the infectious cycle require the hijacking of cellular membranes. Positive stranded RNA viruses replicating in the cytoplasm of their host reorganize cellular membranes. This leads to the formation of organelles, which host viral replication. The formation of such compartments, which are genuine viral factories, induces morphological modifications of the host cell, which vary depending on the pathogen. However, the molecular mechanisms underlying such a remodeling remain unclear. These mechanisms are subject to intense research since their formation is indispensable to viral multiplication and therefore represent an attractive therapeutic target. In this review, we provide a bird's eye view on the current knowledge of the architecture and virus-host interactions involved in the biogenesis of positive stranded RNA virus replication organelles.

Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells.

Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolutionarily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34+ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.

Biology of Zika Virus Infection in Human Skin Cells.

UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.

Vpr expression abolishes the capacity of HIV-1 infected cells to repair uracilated DNA.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein binds to the cellular uracil-DNA glycosylase UNG2 and induces its degradation through the assembly with the DDB1-CUL4 ubiquitin ligase complex. This interaction counteracts the antiviral activity exerted by UNG2 on HIV-1 gene transcription, as previously reported by us. In this work, we show that Vpr expression in the context of HIV-1 infection markedly decreases UNG2 expression in transformed or primary CD4(+) T lymphocytes. We demonstrate for the first time that Vpr-UNG2 interaction significantly impairs the uracil excision activity of infected cells. The loss of uracil excision activity coincides with a significant accumulation of uracilated bases in the genome of infected cells without changes in cell division. Although UNG2 expression and uracil-DNA glycosylase activity are recovered after the peak of retroviral replication, the mutagenic effect of transient DNA uracilation in cycling cells should be taken into account. Therefore, the possible consequences of Vpr-mediated temporary depletion of endogenous nuclear UNG2 and subsequent alteration of the genomic integrity of infected cells need to be evaluated in the physiopathogenesis of HIV infection.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.

Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.

Artificial nucleobase-amino acid conjugates: a new class of TAR RNA binding agents.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is involved in the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop structure, which is encoded by the 5' leader sequence found in all HIV-1 mRNAs. Herein, we report the rational design, synthesis, and in vitro evaluation of new RNA binding agents that were conceived in order to bind strongly and selectively to the stem-loop structure of TAR RNA and, thus, inhibit the Tat/TAR interaction. We have demonstrated that the conjugation of modified nucleobases, able to interact specifically with an RNA base pair, and various amino acids allows these motifs to bind the target RNA selectively and in a cooperative manner that leads to the inhibition of viral replication in HIV-infected cells.

E5564 inhibits immunosuppressive cytokine IL-10 induction promoted by HIV-1 Tat protein.

BACKGROUND: In HIV-1 infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with progression of infection toward AIDS. HIV-1 Tat protein, by interacting with TLR4-MD2 at the membrane level, induces IL-10 production by primary human monocytes and macrophages. In the present study we evaluated the effect of the TLR4 antagonist Eritoran tetrasodium (E5564) on HIV-1 Tat-induced IL-10 production. FINDINGS: Here, we confirm that the recombinant HIV-1 Tat protein and the GST-Tat 1-45 fusion protein efficiently stimulate IL-10 production by primary monocytes and macrophages and that this stimulation is inhibited by blocking anti-TLR4 mAbs. We show that a similar inhibition is observed by preincubating the cells with the TLR4 antagonist E5564. CONCLUSION: This study provides compelling data showing for the first time that the TLR4 antagonist E5564 inhibits the immunosuppressive cytokine IL-10 production by primary human monocytes and macrophages incubated in the presence of HIV-1 Tat protein.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

The virus responsible for COVID-19 infections is known as SARS-CoV-2. Like all viruses, SARS-CoV-2 carries instructions to make proteins and other molecules that play essential roles in enabling the virus to multiply and spread. Viruses are unable to make these molecules themselves, so they infect cells and trick them into making the molecules and assembling new virus particles on their behalf instead. When SARS-CoV2 infects cells, the host cells are reprogrammed to make chains containing several virus proteins that need to be severed from each other by a virus enzyme, known as Nsp5, to enable the proteins to work properly. Previous studies suggested that Nsp5 may also interact with a human protein known as TRMT1, which helps with the production of new proteins in cells. However, it was not clear how Nsp5 may bind to TRMT1 or how this interaction may affect the host cell. Zhang et al. used biochemical and molecular techniques in human cells to study how Nsp5 interacts with TRMT1. The experiments found that the virus enzyme cuts TRMT1 into fragments that are inactive and are subsequently destroyed by the cells. Moreover, Nsp5 cuts TRMT1 at exactly the same position corresponding to the cleavage sites of the viral proteins. Mutation of the sequence in TRMT1 renders Nsp5 ineffective at cutting the protein. SARS-CoV-2 infection caused TRMT1 levels to decrease inside the cells, in turn, leading to a drop in TRMT1 activity. The virus multiplied less in cells that were unable to produce TRMT1 compared to normal human cells, suggesting that the virus benefits from TRMT1 early during infection, before inactivating it at a later point. These findings suggest that one way SARS-CoV-2 causes disease is by decreasing the levels of a human protein that regulates protein production. In the future, the work of Zhang et al. may provide new markers for detecting infections of SARS-CoV-2 and other similar viruses and guide efforts to make more effective therapies against them.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription.

BACKGROUND: Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle. RESULTS: NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions. CONCLUSIONS: Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.

Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following Infection with Dengue Virus.

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

Identification of a non-canonical G3BP-binding sequence in a Mayaro virus nsP3 hypervariable domain.

Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3.

Cellular kinases incorporated into HIV-1 particles: passive or active passengers?

Phosphorylation is one of the major mechanisms by which the activities of protein factors can be regulated. Such regulation impacts multiple key-functions of mammalian cells, including signal transduction, nucleo-cytoplasmic shuttling, macromolecular complexes assembly, DNA binding and regulation of enzymatic activities to name a few. To ensure their capacities to replicate and propagate efficiently in their hosts, viruses may rely on the phosphorylation of viral proteins to assist diverse steps of their life cycle. It has been known for several decades that particles from diverse virus families contain some protein kinase activity. While large DNA viruses generally encode for viral kinases, RNA viruses and more precisely retroviruses have acquired the capacity to hijack the signaling machinery of the host cell and to embark cellular kinases when budding. Such property was demonstrated for HIV-1 more than a decade ago. This review summarizes the knowledge acquired in the field of HIV-1-associated kinases and discusses their possible function in the retroviral life cycle.

Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

BACKGROUND: Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. RESULTS: We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. CONCLUSION: These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.