Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2–5% develop ATL. The past 35 years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-κB pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Background: The rate of transcription of the HIV-1 viral genome is mediated by the interaction of the viral protein Tat with the LTR and other transcriptional machinery. These specific interactions can be affected by the state of post-translational modifications on Tat. Previously, we have shown that Tat can be phosphorylated and acetylated in vivo resulting in an increase in the rate of transcription. In the present study, we investigated whether Tat could be methylated on lysine residues, specifically on lysine 50 and 51, and whether this modification resulted in a decrease of viral transcription from the LTR. Results: We analyzed the association of Tat with histone methyltransferases of the SUV39-family of SET domain containing proteins in vitro. Tat was found to associate with both SETDB1 and SETDB2, two enzymes which exhibit methyltransferase activity. siRNA against SETDB1 transfected into cell systems with both transient and integrated LTR reporter genes resulted in an increase in transcription of the HIV-LTR in the presence of suboptimal levels of Tat. In vitro methylation assays with Tat peptides containing point mutations at lysines 50 and 51 showed an increased incorporation of methyl groups on lysine 51, however, both residues indicated susceptibility for methylation. Conclusion: The association of Tat with histone methyltransferases and the ability for Tat to be methylated suggests an interesting mechanism of transcriptional regulation through the recruitment of chromatin remodeling proteins to the HIV-1 promoter.

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host's miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.

The development of novel techniques and systems to study human infectious diseases in both anin vitroandin vivosettings is always in high demand. Ideally, small animal models are the most efficient method of studying human afflictions. This is especially evident in the study of the human retroviruses, HIV-1 and HTLV-1, in that current simian animal models, though robust, are often expensive and difficult to maintain. Over the past two decades, the construction of humanized animal models through the transplantation and engraftment of human tissues or progenitor cells into immunocompromised mouse strains has allowed for the development of a reconstituted human tissue scaffold in a small animal system. The utilization of small animal models for retroviral studies required expansion of the early CB-17scid/scidmouse resulting in animals demonstrating improved engraftment efficiency and infectivity. The implantation of uneducated human immune cells and associated tissue provided the basis for the SCID-hu Thy/Liv and hu-PBL-SCID models. Engraftment efficiency of these tissues was further improved through the integration of the non-obese diabetic (NOD) mutation leading to the creation of NODSCID, NOD/Shi-scid IL2rγ^-/-, and NOD/SCID β2-microglobulin^nullanimals. Further efforts at minimizing the response of the innate murine immune system produced the Rag2^-/-γ_c^-/-model which marked an important advancement in the use of human CD34+ hematopoietic stem cells. Together, these animal models have revolutionized the investigation of retroviral infectionsin vivo.