Abstract

The ability to trigger an innate immune response against opportunistic pathogens associated with HIV-1 infection is an important aspect of AIDS pathogenesis. Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens, but in HIV-1 patients coinfected with opportunistic infections, the regulation of TLR expression has not been studied. In this context, we have evaluated the expression of TLR2 and TLR4 in monocytes, plasmacytoid dendritic cells, and myeloid dendritic cells of HIV-1 patients with or without opportunistic infections. Forty-nine HIV-1-infected individuals were classified according to viral load, highly active antiretroviral therapy (HAART), and the presence or absence of opportunistic infections, and 21 healthy subjects served as controls. Increased expression of TLR2 and TLR4 was observed in myeloid dendritic cells of HIV-1 patients coinfected with opportunistic infections (without HAART), while TLR4 increased in plasmacytoid dendritic cells, compared to both HIV-1 without opportunistic infections and healthy subjects. Moreover, TLR2 expression was higher in patients with opportunistic infections without HAART and up-regulation of TLR expression in HIV-1 patients coinfected with opportunistic infections was more pronounced in dendritic cells derived from individuals coinfected with Mycobacterium tuberculosis. The results indicate that TLR expression in innate immune cells is up-regulated in patients with a high HIV-1 load and coinfected with opportunistic pathogens. We suggest that modulation of TLRs expression represents a mechanism that promotes HIV-1 replication and AIDS pathogenesis in patients coinfected with opportunistic pathogens.

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1–dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

The RNA interference mechanism utilizes short RNA duplexes to either suppress or induce target gene expression. Post-transcriptional regulation mediated by microRNA is an integral component of innate antiviral defense. The magnitude and the efficiency of viral restriction guided by RNA-based defenses, as well as the full physiological implication of host-pathogen engagement, constitute exciting areas of investigation in the biology of non-coding RNAs.

Background

Hepatitis C virus (HCV) infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL).

Methods

HepG2 and Chang Liver (CHL) cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU) of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis.

Results

Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter.

Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates some interferon response genes and acts as a component of cell defense against viral infection.

Conclusion

HCV core protein have opposite effects on the expression of RANTES in different cell types in vitro, possibly reflecting a similar scenario in different microenvironments in vivo.

Background

Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.

Results

Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.

Conclusion

Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.

MicroRNAs play critical role in regulating gene expression. MicroRNA profile of particular cell type bears the signature of cell type specific gene expression. Given that viral pathogens replicate by evading host defenses, research is now focused on the miRNA-regulated genes that critically regulate HIV-1 propagation in human host cells.

Background

The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction.

Results

Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive.

Conclusion

The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.

The human immunodeficiency virus type 1 (HIV-1) Rev and human T-cell leukemia virus type 1 (HTLV-1) Rex proteins are essential for the expression of viral structural proteins and productive infection. Both contain a nuclear export signal (NES) in their C-terminal domain and a nuclear localization signal (NLS) in their N-terminal domain. The NES and NLS are necessary for shuttling between nucleus and cytoplasm and are therefore indispensable for the transport of unspliced and singly spliced viral transcripts. HIV-1 Rev function is restricted in A9 cells, a murine fibroblast cell line, whereas HTLV-1 Rex is functional in these cells. Immunofluorescence studies with RevGFP fusion protein demonstrate normal import and export of Rev in A9 cells. To ascertain which domains of Rev are necessary for the restriction of Rev function in A9 cells, we studied a chimeric construct in which the NES domain of Rev was exchanged with Rex C-terminal amino acids 79 to 95, the Rev1-79/Rex79-95 chimera, which restored Rev function in A9 cells. In addition, overexpression of a truncated Rev containing the Rev C-terminal domain in the presence of wild-type Rev, led to restoration of Rev function in A9 cells. These results suggest that the C-terminal domain of HIV-1 Rev plays an important role in restricting Rev function in murine cells.

Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G2 phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G1. This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G2 and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G2/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-3H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.