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.

CD28 null CD4 T-cell expansions in autoimmune disease suggest a link with cytomegalovirus infection.

Immunosenescence is thought to contribute to the increase of autoimmune diseases in older people. Immunosenescence is often associated with the presence of an expanded population of CD4 T cells lacking expression of CD28 (CD28 null). These highly cytotoxic CD4 T cells were isolated from disease-affected tissues in patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or other chronic inflammatory diseases and their numbers appeared to be linked to disease severity. However, we recently demonstrated that the common herpes virus, cytomegalovirus (CMV), not ageing, is the major driver of this subset of cytotoxic T cells. In this review, we discuss how CMV might potentiate and exacerbate autoimmune disease through the expansion of CD28 null CD4 T cells.

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.

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).

Genetic and functional characterization of human immunodeficiency virus type 1 VprC variants from north India: presence of unique recombinants with mosaic genomes from B, C and D subtypes within the open reading frame of Vpr.

The human immunodeficiency virus type 1 (HIV-1) epidemic in India is predominantly caused by genetic subtype C, though other minor subtypes have also been reported. One of the major accessory proteins of HIV-1, namely Vpr, is known to influence key steps in viral replication, cell cycle progression, promoter activation, apoptosis and pathogenesis. Therefore, we carried out a genetic and functional analysis of the Vpr variants from eight HIV-1-infected individuals from north India. The sequence analyses revealed that six of eight samples clustered with ancestral subtype C. Remarkably, five of these showed a conserved and region-specific L64P mutation, located in the predicted third alpha-helix. This change adversely affected their ability to activate the HIV-1 long terminal repeat promoter without compromising their ability to cause apoptosis. Bootscan, phylogenetic and SimPlot analysis of the remaining two samples (VprS2 and A6) revealed very interesting mosaic genomes derived from B, C and D subtypes. The N-terminal half of the VprS2 gene consisted of genomic segments derived from subtypes B/D, C and D but the C-terminal half was derived predominantly from subtype C. Interestingly the N-terminal half of sample A6 also showed similar B/D, C and D inter-subtype recombinant structure but the C-terminal half was entirely derived from the consensus B subtype. Multiple breakpoints in a short stretch of 291 nt encoding the Vpr gene strongly suggest that this region is a potential hot-spot for the formation of inter-subtype recombinants and also highlight the importance of the rapidly evolving HIV-1 epidemic in the north Indian region due to multiple genetic subtypes.

Potent knockdown of the X RNA of hepatitis B by a novel chimeric siRNA-ribozyme construct and modulation of intracellular target RNA by selectively disabled mutants.

A multitarget approach is needed for effective gene silencing that combines more than one antiviral strategy. With this in mind, we designed a wild-type (wt) and selectively disabled chimeric mutant (mt) constructs that consisted of small hairpin siRNA joined by a short intracellular cleavable linker to a known hammerhead ribozyme, both targeted against the full-length X RNA of hepatitis B. These chimeric RNAs possessed the ability to cleave the target RNA under in vitro conditions and were efficiently processed at the cleavable site. When this wt chimeric RNA construct was introduced into a liver-specific mammalian cell line, HepG2, along with the HBx substrate encoding DNA, very significant (approximately 70%) intracellular downregulation in the levels of target RNA was observed. When the siRNA portion of this chimeric construct was mutated, keeping the ribozyme (Rz) region unchanged, it caused only approximately 25% intracellular reduction. On the contrary, when only the Rz was made catalytically inactive, about 55% reduction in the target RNA was observed. Construct possessing mt Rz and mt siRNA caused only 10% reduction. This wt chimeric construct also resulted in almost complete knockdown of intracellular HBx protein production, and the mt versions were less effective. The intracellular reduction of target RNA with either wt or mt constructs also interfered with the known functions of HBx protein with varying efficiencies. Thus, in this proof of concept study we show that the levels of the target RNA were reduced potently by the wt chimeric siRNA-Rz construct, which could be modulated with mt versions of the same.

HIV-1 VprB and C RNA cleavage by potent 10-23 DNAzymes that also cause reversal of G2 cell cycle arrest mediated by Vpr genes.

Accessory Vpr protein of HIV-1 is known to influence several key cellular functions that also impacts on the HIV-1 replication cycle. Besides other activities, it alone causes cell cycle arrest at the G2 phase and thus potentially contribute to the overall pathology. We designed several 10-23 catalytic motifs containing DNAzymes (Dzs) against the full-length Vpr gene from subtype B and checked its activity against VprC gene from one of the Indian HIV-1 isolates. Among several Dzs that showed sequence-specific cleavage activities, Dz-94 was very potent and equally efficient in its ability to cleave full-length VprB and C RNA to completion under standard conditions of cleavage. Although Dz-90 target sequence was fully conserved between VprB and C genes, it was more effective on latter genes, suggesting that spatial structures of RNA at other regions of Vpr can also influence the cleavage activity for this Dz. HIV-1 VprB and C encoding genes under the powerful CMV promoter, when cotransfected into mammalian cells with Dz-94, a potent intracellular inhibition, was observed, which also resulted in reversing the G2 cell cycle arrest mediated by VprB and C proteins. Thus, Dz-94 could potentially be developed to prevent Vpr-mediated cytopathic effects caused by HIV-1 subtype B and C isolates.

DNA-enzyme-mediated cleavage of human immunodeficiency virus type 1 Gag RNA is significantly augmented by antisense-DNA molecules targeted to hybridize close to the cleavage site.

DNA-enzymes (Dzs) usually cleave short synthetic target RNAs very efficiently, but this activity diminishes significantly when tested on full-length RNAs, primarily because of the rigid secondary structures near the target sequence. We identified two Dzs, one each for 81-17 and 10-23 Dz, which cleaved the human immunodeficiency virus type 1 (HIV-1) Gag RNA poorly. We sought to use short oligodeoxynucleotides (ODNs) with the hope that it will facilitate Dz-mediated cleavage. The efficiencies of several ODNs were analyzed for their ability to augment the 8-17 Dz-mediated cleavage. We observed that ODNs that hybridized close to 5' and 3' ends of the target sequence were able to enhance significantly 8-17 Dz-mediated cleavage activity in a dose-dependent manner. The same was true for 10-23 Dz with ODNs that hybridized close to the target site. Thus, it was possible to enhance significantly the cleavage activity of poorly cleaving HIV-1 Gag-specific Dzs by using sequence-specific ODNs. This combination of antisense and catalytic Dz will, in principle, result in more effective gene suppression that could be exploited for therapeutic purposes.