Potent intracellular knock down of hepatitis B virus X RNA by catalytic hammerhead ribozymes or DNA-Enzymes with antisense DNA-oligonucleotides or 10-23 DNA-Enzymes that powerfully augment in vitro sequence-specific cleavage activities

Novel antiviral approaches are needed to control Hepatitis B virus infection worldwide. X protein of this virus activates various promoters and is strongly associated with hepatocellular carcinoma. Although several groups, including ours, reported sequence-specific cleavage of X RNA by either ribozymes (Rzs) or DNA-enzymes (Dzs) earlier, but none of these studies reported 100% in vitro cleavage of the full-length X RNA. We reasoned that by melting the secondary structures near the Rz/Dz cleavage site with specific antisense DNA oligonucleotides (ODNs) or 10-23 Dz, it may be possible to achieve this objective. Hammerhead motif containing Rz-170 specific for X RNA was constructed by recombinant techniques and Dz-237 was synthesized using the 10-23 catalytic motif. When specific ODNs or 10-23 Dzs were included in the cleavage reaction with either Rz-170 or Dz-237, increased cleavage was observed in a dose-dependent manner which often resulted in almost complete in vitro cleavage of the target RNA. Rz-170 in combination with specific ODNs caused potent intracellular reduction of HBx RNA. Thus, the cleavage activity of catalytic nucleic acids (Rzs or Dzs) can be increased significantly by specific ODNs or Dzs and this treatment also results in potent intracellular target RNA reduction. These findings have important therapeutic implications

The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins

A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence

In-depth profiling of T-cell responsiveness to commonly recognized CMV antigens in older people reveals important sex differences

The impact of biological sex on T-cell immunity to Cytomegalovirus (CMV) has not been investigated in detail with only one published study comparing CMV-specific T-cell responses in men and women. Many studies, however, have shown an association between CMV infection and immunosenescence, with broad effects on peripheral blood lymphocyte subsets as well as the T and B-cell repertoires. Here, we provide a detailed analysis of CMV-specific T-cell responses in (n=94) CMV+ older people, including 47 women and 47 men aged between 60 and 93 years. We explore sex differences with respect to 16 different CMV proteins arranged in 14 peptide pools (overlapping peptides). Following ex vivo stimulation, CD4 and CD8 T-cells producing IFN-γ, TNF, and IL-2 were enumerated by flow-cytometry (intracellular cytokine staining). T-cell responses were evaluated in terms of each cytokine separately or in terms of cytokines produced simultaneously (polyfunctionality). Surface memory phenotype and CD3 downmodulation were assessed in parallel. The polyfunctionality index and a memory subset differentiation score were used to identify associations between response size, cytokine production, polyfunctionality, and memory subset distribution. While no significant sex differences were found with respect to overall CMV target protein selection, the T-cell response in men appeared more focused and accompanied by a more prominent accumulation of CMV-specific memory CD4 and CD8 T-cells. T-cell polyfunctionality and differentiation were similar in the sexes, however, CMV-specific T-cells in men produced more pro-inflammatory cytokines. Particularly, TNF production by CD4 T-cells was stronger in men than in women. Also, compared with women, men had larger responses to CMV proteins with immediate-early/early kinetics than women, which might have been driven by CMV reactivation. In conclusion, the CMV-specific T-cell response in men was larger and more pro-inflammatory than in women. Our findings may help explain sex differences in CMV-associated pathologies

Cytomegalovirus infection is associated with an increase in aortic stiffness in older men which may be mediated in part by CD4 memory T-cells

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets.Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required.Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people.Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required

The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR)

Nef blocks the sorting of GW182 into exosomes.

<p>Exosomes were purified from the spent culture media of the two cell lines as detailed in Materials and Methods. (A) Western blotting was done using 40 µg of cellular and exosomal lysates using antibodies that detect marker proteins for exosomes (Alix, Tsg101, CD81), mitochondria (VDAC), endoplasmic reticulum (Calnexin) and apoptotic bodies (Cytochrome C). GAPDH and Actin were used as loading controls and anti-GFP antibodies were used to detect Nef-EYFP and EYFP. (B) Cellular and exosomal lysates were subjected to SDS-PAGE and western blotting to detect GW182, Ago2 and Nef; GAPDH was used as a loading control.</p

Characterization of Nef-expressing cell lines.

<p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established as described in Materials and Methods. (A) Western blot for expression of EYFP (lanes Y6-A to -D) and Nef-EYFP (lanes NY-19 and -20) in selected U937 stable clones. (B) Flow cytometric analysis of U937/Nef-EYFP and U937/EYFP cells for surface expression of the indicated proteins. (C) The U937/Nef-EYFP cells were (i) cultured with 5 µM NRhPE, or (ii) stained with anti-Ago2 as described in Materials and Methods. U1 cells were activated with PMA and labeled with anti-Nef and either (iii) anti-Ago2 or (iv) anti-P bodies as described in Materials and Methods. The white arrowheads in the merged image represent colocalization points. The images are representative of three independent experiments. The boxed regions are expanded. (D) Correlation analysis of the colocalization of Nef with Ago2 in (i) U937/Nef-EYFP cells and (ii) activated U1 cells showing Pearson’s coefficient (PC) and Manders coefficients (M1, M2). The coefficients represent an average of three independent images, each consisting of at least 10 cells, calculated using the JACoP plugin within the Image J software. M1 represents the fraction of Ago2-red overlapping with Nef-green and M2 represents the fraction of Nef-green overlapping with Ago2- red. Bars represent ± SD.</p

Mutations in Nef GW motifs abrogate its interaction with Ago2 and reduce virus yield and infectivity.

<p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los Alamos HIV database were analyzed. Regions encompassing the two conserved GW motifs (bold and underline) at positions 12–13 and 140–141 are shown for consensus and ancestral (.anc) sequences for various HIV-1 M group clades (A–H), and consensus sequences for various recombinant circulating forms. (B) HEK293T cells were transiently transfected to express wild type or GW mutant Nef proteins. Cell lysates were precipitated with anti-Nef and blotted with anti-Ago2. Nef and Actin were expression and loading controls, respectively. (C) HEK293T cells were transfected with either pNL4-3 or pNL4-3ΔNef; cells were also co-transfected with pNL4-3ΔNef and expression vectors for either the wild type or GW mutant Nef proteins. After 36 hr the culture supernatants were quantified for p24. Purified viruses equivalent to 100ng p24 were also used to infect 0.5 million 1G5 Jurkat indicator cells, and 48 hr later the cells were harvested, lysed and luciferase assay was performed as described in Methods. Error bars represent mean ± SD from three independent experiments, and p-values calculated using the Student’s <i>t</i>-test were as follows: * 0.002; # 0.023.</p

Nef interacts with Ago2.

<p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. The lane marked NIC represents IP of U937/Nef-EYFP lysates with an irrelevant antibody. (B) U1 and J1.1 cells were treated with DMSO (–) or PMA (+) and cell lysates were either immunoblotted for p24 and Nef (i, ii), or IP/IB with anti-Nef and anti-Ago2 (iii, iv), as described in Materials and Methods. U937 and Jurkat cells served as background controls and antibody heavy chain as loading control. (C) Lysates were prepared from the U937 stable cell lines using Buffer D as described in Materials and Methods. From this, 150 µg lysate was passed through a pre-equilibrated Sephacryl S200HR column with a 3 ml bed volume. The column was eluted and fractions of 3 drops (∼120 µl) were collected, which were then precipitated with acetone, separated by SDS-PAGE and western blotting was done with Ago2 and GFP antibodies. (D) Lysates from activated U1 cells were prepared and fractionated as in (C) followed by SDS-PAGE and western blotting with Ago2 and Nef antibodies. The profiles shown are representative of three separate experiments.</p

Nef does not affect siRNA loading into RISC, but inhibits its slicing activity.

<p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Materials and Methods. From this, 25 µg of lysate was incubated with ³²P-labeled duplex siRNA (without or with 50-fold molar excess of unlabeled siRNA) in binding buffer for 30 min at 4°C. The complexes were separated on a 6% non-denaturing polyacrylamide gel. (B) The gel shift assay was set up as in (A) except that lysates immunodepleted for either Ago2 or Dicer were also included. The positions of the mobility shifted complexes and free probe are indicated. (C) Slicer activity was assayed using immunoprecipitated Ago2 from U937/Nef-EYFP or U937/EYFP cell lysates and a ³²P-labeled <i>in vitro</i> transcribed let7a RNA, as described in Materials and Methods. The positions of full-length RNA and the two sliced products are shown. The 59 nt marker oligonucleotide was run on the same gel, but a lower exposure is shown. (D) Densitometric analysis of autoradiograms from three independent experiments was carried out using Image J (version 1.4.1). The slicing activity in Nef-EYFP lysates is represented as a percentage of that in EYFP lysates.</p