Schlafen 11 Restricts Flavivirus Replication.

Schlafen 11 (Slfn11) is an interferon-stimulated gene that controls the synthesis of proteins by regulating tRNA abundance. Likely through this mechanism, Slfn11 has previously been shown to impair human immunodeficiency virus type 1 (HIV-1) infection and the expression of codon-biased open reading frames. Because replication of positive-sense single-stranded RNA [(+)ssRNA] viruses requires the immediate translation of the incoming viral genome, whereas negative-sense single-stranded RNA [(-)ssRNA] viruses carry at infection an RNA replicase that makes multiple translation-competent copies of the incoming viral genome, we reasoned that (+)ssRNA viruses will be more sensitive to the effect of Slfn11 on protein synthesis than (-)ssRNA viruses. To evaluate this hypothesis, we tested the effects of Slfn11 on the replication of a panel of ssRNA viruses in the human glioblastoma cell line A172, which naturally expresses Slfn11. Depletion of Slfn11 significantly increased the replication of (+)ssRNA viruses from the Flavivirus genus, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), but had no significant effect on the replication of the (-)ssRNA viruses vesicular stomatitis virus (VSV) (Rhabdoviridae family) and Rift Valley fever virus (RVFV) (Phenuiviridae family). Quantification of the ratio of genome-containing viral particles to PFU indicated that Slfn11 impairs WNV infectivity. Intriguingly, Slfn11 prevented WNV-induced downregulation of a subset of tRNAs implicated in the translation of 11.8% of the viral polyprotein. Low-abundance tRNAs might promote optimal protein folding and enhance viral infectivity, as previously reported. In summary, this study demonstrates that Slfn11 restricts flavivirus replication by impairing viral infectivity.IMPORTANCE We provide evidence that the cellular protein Schlafen 11 (Slfn11) impairs replication of flaviviruses, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV). However, replication of single-stranded negative RNA viruses was not affected. Specifically, Slfn11 decreases the infectivity of WNV potentially by preventing virus-induced modifications of the host tRNA repertoire that could lead to enhanced viral protein folding. Furthermore, we demonstrate that Slfn11 is not the limiting factor of this novel broad antiviral pathway.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

BACKGROUND: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. RESULTS: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. CONCLUSIONS: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity.

Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4(+) T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4(+) T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors.

Poly(ADP-ribose) polymerase-1 silences retroviruses independently of viral DNA integration or heterochromatin formation.

PARP-1 silences retrotransposons in Drosophila, through heterochromatin maintenance, and integrated retroviruses in chicken. Here, we determined the role of viral DNA integration and cellular heterochromatin in PARP-1-mediated retroviral silencing using HIV-1-derived lentiviral vectors and Rous-associated virus type 1 (RAV-1) as models. Analysis of the infection of PARP-1 knockout and control cells with HIV-1 harbouring WT integrase, in the presence or absence of an integrase inhibitor, or catalytic-dead mutant integrase indicated that silencing does not require viral DNA integration. The mechanism involves the catalytic activity of histone deacetylases but not that of PARP-1. In contrast to Drosophila, lack of PARP-1 in avian cells did not affect chromatin compaction globally or at the RAV-1 provirus, or the cellular levels of histone H3 N-terminal acetylated or Lys27 trimethylated, as indicated by micrococcal nuclease accessibility and immunoblot assays. Therefore, PARP-1 represses retroviruses prior to viral DNA integration by mechanisms involving histone deacetylases but not heterochromatin formation.

Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

Poly(ADP-ribose) polymerase 1 promotes transcriptional repression of integrated retroviruses.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a cellular enzyme with a fundamental role in DNA repair and the regulation of chromatin structure, processes involved in the cellular response to retroviral DNA integration. However, the function of PARP-1 in retroviral DNA integration is controversial, probably due to the functional redundancy of the PARP family in mammalian cells. We evaluated the function of PARP-1 in retroviral infection using the chicken B lymphoblastoid cell line DT40. These cells lack significant PARP-1 functional redundancy and efficiently support the postentry early events of the mammalian-retrovirus replication cycle. We observed that DT40 PARP-1(-/-) cells were 9- and 6-fold more susceptible to infection by human immunodeficiency virus type 1 (HIV-1)- and murine leukemia virus (MLV)-derived viral vectors, respectively, than cells expressing PARP-1. Production of avian Rous-associated virus type 1 was also impaired by PARP-1. However, the susceptibilities of these cell lines to infection by the nonretrovirus vesicular stomatitis virus were indistinguishable. Real-time PCR analysis of the HIV-1 life cycle demonstrated that PARP-1 did not impair reverse transcription, nuclear import of the preintegration complex, or viral DNA integration, suggesting that PARP-1 regulates a postintegration step. In support of this hypothesis, pharmacological inhibition of the epigenetic mechanism of transcriptional silencing increased retroviral expression in PARP-1-expressing cells, suppressing the differences observed. Further analysis of the implicated molecular mechanism indicated that PARP-1-mediated retroviral silencing requires the C-terminal region, but not the enzymatic activity, of the protein. In sum, our data indicate a novel role of PARP-1 in the transcriptional repression of integrated retroviruses.

Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner.

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that, in a variety of cell types, including primary immune cells, SLFN14 regulates the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibits the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal RNA degradation is involved. Therefore, we propose that SLFN14 impairs the expression of HIV-1 transcripts rich in rare codons, in a catalytic-dependent manner.

Poly (ADP-ribose) polymerase-1 regulates HIV-1 replication in human CD4+ T cells.

The cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) regulates multiple processes that are potentially implicated in HIV-1 infection. However, the role of PARP-1 in HIV-1 infection remains controversial, with reports indicating or excluding that PARP-1 influence early steps of the HIV-1 life cycle. Most of these studies have been conducted with Vesicular Stomatitis virus Glycoprotein G (VSV-G)-pseudotyped, single-round infection HIV-1; limiting our understanding of the role of PARP-1 in HIV-1 replication. Therefore, we evaluated the effect of PARP-1 deficiency or inhibition in HIV-1 replication in human CD4+ T cells. Our data showed that PARP-1 knockout increased viral replication in SUP-T1 cells. Similarly, a PARP-1 inhibitor that targets PARP-1 DNA-binding activity enhanced HIV-1 replication. In contrast, inhibitors affecting the catalytic activity of the enzyme were inactive. In correspondence with the pharmacological studies, mutagenesis analysis indicated that the DNA-binding domain was required for the PARP-1 anti-HIV-1 activity, but the poly-ADP-ribosylation activity was dispensable. Our results also demonstrated that PARP-1 acts at the production phase of the viral life cycle since HIV-1 produced in cells lacking PARP-1 was more infectious than control viruses. The effect of PARP-1 on HIV-1 infectivity required Env, as PARP-1 deficiency or inhibition did not modify the infectivity of Env-deleted, VSV-G-pseudotyped HIV-1. Furthermore, virion-associated Env was more abundant in sucrose cushion-purified virions produced in cells lacking the enzyme. However, PARP-1 did not affect Env expression or processing in the producer cells. In summary, our data indicate that PARP-1 antagonism enhances HIV-1 infectivity and increases levels of virion-associated Env. Importance: Different cellular processes counteract viral replication. A better understanding of these interfering mechanisms will enhance our ability to control viral infections. We have discovered a novel, antagonist effect of the cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) in HIV-1 replication. Our data indicate that PARP-1 deficiency or inhibition augment HIV-1 infectivity in human CD4+ T cells, the main HIV-1 target cell in vivo . Analysis of the mechanism of action suggested that PARP-1 antagonism increases in the virus the amounts of the viral protein mediating viral entry to the target cells. These findings identify for the first time PARP-1 as a host factor that regulates HIV-1 infectivity, and could be relevant to better understand HIV-1 transmission and to facilitate vaccine development.

[Nutrient dynamics in forest plantations of Azadirachta indica (Meliaceae) established for restoration of degraded lands in Colombia]. / Dinámica de nutrientes en plantaciones forestales de Azadirachta indica (Meliaceae) establecidas para restauración de tierras degradadas en Colombia.

Nutrient dynamics in forest plantations of Azadirachta indica (Meliaceae) established for restoration of degraded lands in Colombia. Azadirachta indica is a tree species which use is steadily increasing for restoration of tropical and subtropical arid and degraded lands throughout the world. The objective of this research study was to evaluate the potential of these plantations as an active restoration model for the recovery of soils under desertification in arid lands of Colombia. Litter traps and litter-bags were installed in twenty 250m2 plots. Green leaves and soil samples inside and outside this species plantations were taken, and their elemental concentrations were determined. Litterfall, leaf litter decomposition and foliar nutrient resorption were monitored for one year. The annual contributions of organic material, such as fine litterfall, represented 557.54kg/ha, a third of which was A. indica leaves. The greatest potential returns of nutrients per foliar litterfall were from Ca (4.6kg/ha) and N (2.4kg/ha), and the smallest potential returns came from P (0.06kg/ha). A total of 68% of the foliar material deposited in litter-bags disappeared after one year. The greatest release of nutrients was that of K (100%), and the least was that of N (40%). P was the most limiting nutrient, with low edaphic availability and high nutrient use efficiency from Vitousek's index (IEV = 3176) and foliar nutrient resorption (35%). Despite these plantations are young, and that they have not had forestry management practices, as an active restoration model, they have revitalized the biogeochemical cycle, positively modifying the edaphic parameters according to the increases in organic material, P and K of 72%, 31% and 61%, respectively. Furthermore, they improved the stability of aggregates and the microbe respiration rates. The forest plantation model with exotic species has been opposed by different sectors; however, it has been acknowledged that these projects derive many benefits for the restoration of biodiversity and ecosystemic functions. The conditions of severe land degradation demand the initial use of species, such as A. indica, that can adapt quickly and successfully, and progressively reestablish the biogeochemical cycle.

A role for LEDGF/p75 in targeting HIV DNA integration.

HIV DNA integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and might therefore direct integration by a tethering interaction. We analyzed HIV integration in cells depleted for LEDGF/p75, and found that integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells.

Correction: Krízová et al. Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein. Viruses 2021, 13, 2451.

The authors wish to make the following corrections to this paper [...].

HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin.

BACKGROUND: Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. RESULTS: Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD). CONCLUSIONS: Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

Implication of serine residues 271, 273, and 275 in the human immunodeficiency virus type 1 cofactor activity of lens epithelium-derived growth factor/p75.

Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular cofactor for HIV-1 DNA integration. It is well established that the simultaneous binding of LEDGF/p75 to chromatin and to HIV-1 integrase is required for its cofactor activity. However, the exact molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet completely understood. Our hypothesis is that evolutionarily conserved regions in LEDGF/p75 exposed to solvent and harboring posttranslational modifications may be involved in its HIV-1 cofactor activity. Therefore, a panel of LEDGF/p75 deletion mutants targeting these protein regions were evaluated for their HIV-1 cofactor activity, chromatin binding, integrase interaction, and integrase-to-chromatin-tethering activity by using different cellular and biochemical approaches. The deletion of amino acids 267 to 281 reduced the cofactor activity of LEDGF/p75 to levels observed for chromatin-binding-defective mutants. This region contains a serine cluster (residues 271, 273, and 275) recurrently found to be phosphorylated in both human and mouse cells. Importantly, the conversion of these Ser residues to Ala was sufficient to impair the ability of LEDGF/p75 to mediate HIV-1 DNA integration, although these mutations did not alter chromatin binding, integrase binding, or the integrase-to-chromatin-tethering capability of LEDGF/p75. These results clearly indicated that serine residues 271, 273, and 275 influence the HIV-1 cofactor activity of integrase-to-chromatin-tethering-competent LEDGF/p75.

LEDGF/p75 proteins with alternative chromatin tethers are functional HIV-1 cofactors.

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi's sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Virological and cellular roles of the transcriptional coactivator LEDGF/p75.

The chromatin-associated cellular proteins LEDGF/p75 and LEDGF/p52 have been implicated in transcriptional regulation, cell survival and autoimmunity. LEDGF/p75 also appears to act as a chromatin-docking factor or receptor for HIV-1 and other lentiviruses and to play a role in leukemogenesis. For both the viral and cellular roles of this protein, a key feature is its ability to act as a molecular adaptor and tether proteins to the chromatin fiber. This chapter reviews the emerging roles of LEDGF/p75 and LEDGF/p52 in diverse cellular processes and disease states.

Intensive RNAi with lentiviral vectors in mammalian cells.

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.

Rapid, controlled and intensive lentiviral vector-based RNAi.

RNA interference (RNAi) is a powerful technology for studying the functional significance of genes. The technique is more accessible than gene knockout methods, and is directly applicable to diverse human cells. However, inadequate reductions in target mRNAs can reduce the utility of RNAi and insufficiently rigorous controls can lead to spurious conclusions. Optimally combining pol III promoters to drive short hairpin RNA expression with the gene transfer capabilities of lentiviral vectors has led to ways to perform especially effective and convincing RNAi, which we review here. We detail practical methods, including one-step vector construction. Deep, stable knockdowns to trace mRNA levels are readily achieved in T cell lines, which can then be subjected to comprehensive HIV challenge studies. Rescue of preknockdown phenotype by RNAi-resistant gene re-expression is a critical validating step. The methods can also be applied to primary T cells and macrophages. The time from thinking of a target to initial data read-out can be a few weeks.

Correction: A new family of fullerene derivatives: fullerene-curcumin conjugates for biological and photovoltaic applications.

[This corrects the article DOI: 10.1039/C8RA08334G.].

Defining Pharmacological Targets by Analysis of Virus-Host Protein Interactions.

Viruses are obligate parasites that depend on cellular factors for replication. Pharmacological inhibition of essential viral proteins, mostly enzymes, is an effective therapeutic alternative in the absence of effective vaccines. However, this strategy commonly encounters drug resistance mechanisms that allow these pathogens to evade control. Due to the dependency on host factors for viral replication, pharmacological disruption of the host-pathogen protein-protein interactions (PPIs) is an important therapeutic alternative to block viral replication. In this review we discuss salient aspects of PPIs implicated in viral replication and advances in the development of small molecules that inhibit viral replication through antagonism of these interactions.