Explore the vast range of titles available.

N 6 -methyladenosine (m 6 A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m 6 A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m 6 A, respectively. HIV-1 RNA contains m 6 A modifications that modulate viral infection and gene expression in CD4 + T cells. However, it remains unclear whether m 6 A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m 6 A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m 6 A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m 6 A levels of RNA by manipulating the expression of the m 6 A erasers (FTO and ALKBH5) or pharmacological inhibition of m 6 A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro . HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m 6 A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m 6 A modifications had opposite effects. Our mechanistic studies indicated that m 6 A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m 6 A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.

The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear. In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques. We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells. Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.

The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. HIV infection is a global health challenge. The antiviral drugs that are currently available limit the ability of the virus to multiply in infected individuals, but they rarely eliminate the virus entirely. A better understanding of how HIV behaves in the cell would help researchers to find a cure for persistent HIV infection. When HIV enters an immune cell, its genetic material – in the form of molecules of ribonucleic acid (RNA) – is used as a template to make molecules of DNA. This viral DNA can integrate into the host cell’s DNA, where it is used as a template to make more viral RNA molecules, which are then used to make viral proteins. Some of the viral RNAs are also packaged into new virus particles. In cells, RNA molecules are often subject to a chemical modification called adenosine methylation, which regulates how that RNA is translated into proteins. Specific enzymes add molecules called methyl tags to particular locations on the RNA, while other enzymes remove them. A family of proteins called YTHDF1–3 recognize and bind to these methyl tags on the RNA and influence how much protein is produced from the target RNA. There is evidence to suggest that the cell can add methyl tags to HIV RNA. However, the extent to which this happens, and what effects this modification has on HIV replication and viral protein production, are not clear. Tirumuru et al. addressed these questions by analyzing how changing the levels of YTHDF1–3 proteins and the enzymes that add or remove methyl tags in human cells affected HIV infection. The experiments show that YTHDF1–3 proteins inhibited HIV infection in immune cells called T-lymphocytes by recognizing HIV RNA that had been methylated, mainly by targeting the step where the viral RNA is copied into DNA. Altering the levels of the enzymes that add or remove methyl tags in the cells can change the amount of methyl tags attached to RNA molecules, which alters the amount of HIV protein produced. For example, when more RNA molecules had methyl tags, the cells produced more HIV proteins. These findings suggest that adenosine methylation plays an important role in regulating the ability of HIV to thrive and multiply in T-lymphocytes, which are an important target for HIV. Since the RNAs of other human viruses, such as influenza virus, can also be modified by adenosine methylation, drugs that target this pathway could have the potential to be used to fight a variety of viral illnesses.

Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus)-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N) (~120 kDa) inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4(+) T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.

Encephalitis can be caused by viruses, and it is potentially life-threatening because of the vital nature of the brain and the lack of treatment options. MAV-1 produces viral encephalitis in its natural host, providing a model for investigating factors involved in development of encephalitis. MAV-1 infection disrupts the BBB and increases activity of matrix metalloproteinases in brains of infected mice. We investigated whether the major transcriptional regulator of adenoviruses, E1A protein, is responsible for any of the specific phenotypes that result from MAV-1 infection. For some of the functions assayed, an E1A mutant virus behaved like wild-type virus. However, expression of mRNA for one matrix metalloproteinase was higher in the virus lacking E1A protein production. This highlights the complex nature of encephalitis and suggests that E1A may have transcriptional effects on host genes important for the development of encephalitis. Mouse adenovirus type 1 (MAV-1) infects endothelial cells and disrupts the blood-brain barrier (BBB), causing encephalitis in inbred and outbred mice. Using a virus mutant that does not produce the early region 1A protein E1A, we investigated whether the activity of this known viral transcriptional regulator is needed for BBB disruption and other phenotypes associated with encephalitis. The wild-type (wt) virus and E1A mutant virus caused similar levels of permeability of sodium fluorescein in brains of infected mice. In an in vitro assay of BBB integrity, wt and mutant virus caused similar decreases in transendothelial electrical resistance in primary mouse brain endothelial cell monolayers. These results indicate that E1A protein does not contribute to disruption of BBB integrity in animals or cultured cells. Both wt and E1A mutant virus infection of mice led to similar increases in the activity of two matrix metalloproteinases known to correlate with BBB disruption, MMP2 and MMP9, while causing no increase in the steady-state expression of MMP2 or MMP9 mRNA. In contrast, the amount of MMP3 transcripts increased upon infection by both viruses and to a higher level in infections by the mutant virus lacking E1A protein production. There was no difference in the levels of steady-state expression of mRNA for tight junction proteins among mock virus, wt virus, and mutant virus infections. Thus, the MAV-1 E1A protein does not measurably affect BBB integrity in the parameters assayed, although it reduces the amount of MMP3 mRNA steady-state expression induced in brains upon infection. IMPORTANCE Encephalitis can be caused by viruses, and it is potentially life-threatening because of the vital nature of the brain and the lack of treatment options. MAV-1 produces viral encephalitis in its natural host, providing a model for investigating factors involved in development of encephalitis. MAV-1 infection disrupts the BBB and increases activity of matrix metalloproteinases in brains of infected mice. We investigated whether the major transcriptional regulator of adenoviruses, E1A protein, is responsible for any of the specific phenotypes that result from MAV-1 infection. For some of the functions assayed, an E1A mutant virus behaved like wild-type virus. However, expression of mRNA for one matrix metalloproteinase was higher in the virus lacking E1A protein production. This highlights the complex nature of encephalitis and suggests that E1A may have transcriptional effects on host genes important for the development of encephalitis.

Rabies is an important public health problem in South East Asia, with cases in this part of the world contributing to about 70% of the global burden. A large number of rabies cases occur in India, however, there is no organized system of surveillance and hence there is a lack of reliable data. Moreover, comprehensive molecular epidemiological studies have not been performed on Indian virus isolates. In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus obtained from the brain of an infected patient. Comparison of the genomic sequence with those of the ten fully sequenced rabies strains available in GenBank showed nucleotide homology ranging from 97% with AY956319 to 81% with AY705373. Amino acid homology of nucleoprotein ranged from 99.7% with AY352493 to 92% with DQ875051. In case of the glycoprotein gene, the homology ranged from 98.8% with AY956319 to 87.2 % with AY705373. An extensive nucleoprotein, glycoprotein, and full-length genome-based phylogenetic analysis was performed along with sequences available from the GenBank. Phylogenetic analysis of the complete genome sequence indicated that this isolate exhibited close homology with the ex Indian strain AY956319.