ABSTRACT

Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We report here that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs.

IMPORTANCE

Long protein-noncoding RNAs (lncRNAs) play roles in regulating gene expression and modulating protein activities. There is emerging evidence that lncRNAs are involved in the replication of viruses. To our knowledge, this report is the first to characterize a role contributed by an lncRNA, NEAT1, to HIV-1 replication. NEAT1 is essential for the integrity of the nuclear paraspeckle substructure. Based on our findings from NEAT1 knockdown, we have identified the nuclear paraspeckle body as another important subcellular organelle for HIV-1 replication.

Viral replication requires the use of host cell proteins and enzymes. Many viruses utilize viral helicases at various stages of their life cycle; these viruses have evolved to encode directly helicase or helicase-like proteins. In contrast, the genomes of retroviruses are devoid of viral helicases. Human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. In this chapter, we briefly summarize the approach for assaying the RNA unwinding activity of RNA helicasesd and measurement of the effect helicase inhibitors on HIV-1 replication.

The human T – cell leukemia virus type 1 (HTLV-1) was the first retrovirus discovered to be causative of a human cancer, Adult T-cell leukemia (ATL). The transforming entity of HTLV-1 has been attributed to the virally-encoded oncoprotein, Tax. Unlike the v-onc proteins encoded by other oncogenic animal retroviruses that transform cells, Tax does not originate from a c-onc counterpart. In this article, we review progress in our understanding of HTLV-1 infectivity, cellular transformation, anti-sense transcription, and therapy, thirty years after the original discovery of this virus.

The post-transcriptional export of spliced and unspliced HIV-1 (human immunodeficiency virus type 1) RNAs from the nucleus to the cytoplasm is a complex process. Part of the complexity arises from the fact that eukaryotic cells normally retain unspliced RNAs in the nucleus preventing their exit into the cytoplasm. HIV-1 has evolved a protein, Rev, that participates in the export of unspliced/partially spliced viral RNAs from the nucleus. It has been documented that several cellular factors cooperate in trans with Rev, and certain cis-RNA motifs/features are important for transcripts to be recognized by Rev and its co-factors. Here, the post-transcriptional activities of Rev are discussed in the context of a recent finding that an RNA cap methyltransferase contributes to the expression of unspliced/partially spliced HIV-1 transcripts.

Choosing the right laboratory in which to do postdoctoral training is perhaps one of the most important decisions that a scientist makes in his or her career. Does one choose a laboratory based on the research topic or the research style of the mentor? Does one choose a large laboratory or a small one? How does one fit the selection of a postdoctoral laboratory into the context of one’s long-range career goals? Here, I briefly discuss three points worth considering in seeking a research laboratory for postdoctoral training after the completion of a graduate degree.

Human tumor viruses are associated with a variety of human malignancies, and it is estimated that 15% of all human cancers have a viral etiology. An abnormality in chromosomal ploidy or aneuploidy is a hallmark of cancers. In normal cells, euploidy is governed by several factors including an intact spindle assembly checkpoint (SAC), accurate centrosome duplication, and proper cytokinesis. Viral oncoproteins are suggested to perturb the cellular machineries for chromosomal segregation creating aneuploidy which can lead to the malignant transformation of infected cells. Here we review in brief some of the mechanisms employed by viruses that can cause cellular aneuploidy.

The mechanism of HTLV-1 transformation of cells to Adult T cell leukemia (ATL) remains not fully understood. Currently, the viral Tax oncoprotein is known to be required to initiate transformation. Emerging evidence suggests that Tax is not needed to maintain the transformed ATL phenotype. Recent studies have shown that HTLV-1 transformed cells show deregulated expression of cellular microRNAs (miRNAs). Here we discuss the possibility that early ATL cells are Tax-oncogeneaddicted while late ATL cells are oncogenic microRNA (oncomiR) – addicted. The potential utility of interrupting oncomiR addiction as a cancer treatment is broached.

Contributing reviewers

Retrovirology would like to thank the following for their assistance with peer review of manuscripts for the journal in 2012.

Hereditary hearing loss is the most common sensory deficit. We determined that progressive high-frequency hearing loss in 2 families of Iraqi Jewish ancestry was due to homozygosity for the protein truncating mutation SYNE4 c.228delAT. SYNE4, a gene not previously associated with hearing loss, encodes nesprin-4 (NESP4), an outer nuclear membrane (ONM) protein expressed in the hair cells of the inner ear. The truncated NESP4 encoded by the families’ mutation did not localize to the ONM. NESP4 and SUN domain–containing protein 1 (SUN1), which localizes to the inner nuclear membrane (INM), are part of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the nuclear envelope. Mice lacking either Nesp4 or Sun1 were evaluated for hair cell defects and hearing loss. In both Nesp4–/– and Sun1–/– mice, OHCs formed normally, but degenerated as hearing matured, leading to progressive hearing loss. The nuclei of OHCs from mutant mice failed to maintain their basal localization, potentially affecting cell motility and hence the response to sound. These results demonstrate that the LINC complex is essential for viability and normal morphology of OHCs and suggest that the position of the nucleus in sensory epithelial cells is critical for maintenance of normal hearing.

Retroviruses have an intricate life cycle. There is much to be learned from studying retrovirus-host interactions. Among retroviruses, the primate lentiviruses have one of the more complex genome structures with three categories of viral genes: structural, regulatory, and accessory genes. Over time, we have gained increasing understanding of the lentivirus life cycle from studying host factors that support virus replication. Similarly, studies on host restriction factors that inhibit viral replication have also made significant contributions to our knowledge. Here, we review recent progress on the rapidly growing field of restriction factors, focusing on the antiretroviral activities of APOBEC3G, TRIM5, tetherin, SAMHD1, MOV10, and cellular microRNAs (miRNAs), and the counter-activities of Vif, Vpu, Vpr, Vpx, and Nef.

The importance of geographic diversity in publishing is emphasized in this editorial.

Human T-cell Leukemia Virus type 1 (HTLV-1) and 2 (HTLV-2) are two closely related human retroviruses. HTLV-1 is associated with an aggressive Adult T-cell Leukemia (ATL) while there is no evidence for an association of HTLV-2 with any human malignancies. The two viruses encode transactivator proteins, Tax-1 and Tax-2 respectively. In ATL, Tax-1 is thought to play a central role in the transformation of a normal T-cell into a leukemic cell; however, it has not been entirely clear how post-translational modifications of Tax-1 influence its transforming activity. Here, we discuss three recent papers that report on the ubiquitination and sumoylation of Tax-1 and Tax-2. We comment on their divergent findings implicating the importance (or lack of importance) of these modifications and other events on Tax activation of NF-κB as related to cellular transformation.

Background

Tombusvirus P19 is a protein encoded by tomato bushy stunt virus and related tombusviruses. Earlier studies have demonstrated that P19 is an RNA silencing suppressor (RSS) in plant cells. However, it has not been systematically investigated how P19 suppresses RNA interference in various mammalian cell settings.

Results

We have studied the RSS effect of P19 in mammalian cells, HEK293T, HeLa, and mouse embryonic fibroblasts. We have individually mutated 18 positively charged residues in P19 and found that 6 of these charged residues in P19 reduce its ability to suppress RNA interference. In each case, the reduction of silencing of RNA interference correlated with the reduced ability by these P19 mutants to bind siRNAs (small interfering RNAs).

Conclusions

Our findings characterize a class of RNA-binding proteins that function as RSS moieties. We find a tight correlation between positively charged residues in P19 accounting for siRNA-binding and their RSS activity. Because P19’s activity is conserved in plant and animal cells, we conclude that its RSS function unlikely requires cell type-specific co-factors and likely arises from direct RNA-binding.

Recent literature highlights at Retrovirology are described. Predictions are made regarding “hot” retrovirology research trends for the coming year based on recent journal access statistics. Changes in Retrovirology editor and the frequency of the Retrovirology Prize are announced.

Summary

MicroRNAs (miRNAs) are small non-coding RNAs that control a multitude of critical processes in mammalian cells. Increasing evidence has emerged that host miRNAs serve in animal cells to restrict viral infections. In turn, many viruses encode RNA silencing suppressors (RSS) which are employed to moderate the potency of the cell’s miRNA selection against viral replication. Some viruses also encode viral miRNAs. In this review, we summarize findings from human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) that illustrate examples of host cell miRNAs that target the viruses, of RSS encoded by viruses, and of host cell miRNA profile changes that are seen in infected cells.

Autophagy is a protein degradative process important for normal cellular metabolism. It is apparently used also by cells to eliminate invading pathogens. Interestingly, many pathogens have learned to subvert the cell’s autophagic process. Here, we review the interactions between viruses and cells in regards to cellular autophagy. Using findings from hepatitis B virus and human retroviruses, HIV-1 and HTLV-1, we discuss mechanisms used by viruses to usurp cellular autophagy in ways that benefit viral replication.

Cell and Bioscience is on track to receive its first Impact Factor in mid-2013. What is the role of the Impact Factor as a measure of a journal’s success?

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

Nine years after its founding, Retrovirology has moved to the forefront of virology journals in Impact Factor.

With the recent development of siRNA and shRNA expression libraries, RNAi technology has been extensively employed to identify genes involved in diverse cellular processes, such as signal transduction, cell cycle, cancer biology and host-pathogen interactions. In the field of viral infection, this approach has already identified hundreds of new genes not previously known to be important for various virus lifecycles. In this brief review, we focus on recent studies performed using genome-wide RNAi-based screens in mammalian cells for the identification of essential host factors for viral infection and pathogenesis.

MicroRNAs (miRNAs) are small physiological non-coding RNAs that regulate gene expression through an RNA interference (RNAi) mechanism. The expression of miRNAs is tightly controlled both spatially and temporally. Aberrant miRNA expression has been correlated with various cancers. Recent findings suggest that some miRNAs can function as tumor suppressors or oncogenes. In model experiments, the cancer phenotype of some cells can be reverted to normal when the cells are treated with miRNA mimics or inhibitors. Here, we discuss in brief the potential utility of miRNA-based cancer therapy as well as the current limitations thwarting their useful clinical application.

Although RNA interference (RNAi) is known to play an important part in defense against viruses of invertebrates, its contribution to mammalian anti-viral defense has been a matter of dispute. This is surprising because all components of the RNAi machinery necessary for robust RNAi-mediated restriction of viruses are conserved in mammals, and the introduction of synthetic small interfering RNAs (siRNAs) into cells efficiently silences the replication of viruses that contain siRNA complementary sequences in those cells. Here, I discuss the reasons for the dispute, and review the evidence that RNAi is a part of the physiological defense of mammalian cells against viral infections.

Background

Expression of the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein rapidily induces a significant increase of micronuclei (MN) and unstabilized DNA breaks in cells. Unstabilized DNA breaks can have free 3′-OH ends accessible to in situ addition of digoxygenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. In the present work, we used a GFP-Tax (green fluorescent protein) plasmid, which produces a functionally active GFP-tagged Tax protein, to detect the cellular target(s) for Tax which might mechanistically explain the clastogenic phenomenon. We examined the induction of MN and unstabilized DNA breaks in wild type cells and cells individually knocked out for Ku80, PKcs, XRCC4, and H2AX proteins. We also assessed in the same cells, the signal strengths produced by DIG-dUTP incorporation at the unstable DNA breaks in the presence and absence of Tax.

Results

Cells mutated for PKcs, XRCC4 and H2AX showed increased frequency of MN and unstabilized DNA breaks in response to the expression of Tax, while cells genetically mutated for Ku80 were refractory to Tax’s induction of these cytogenetic effects. Moreover, by measuring the size of DIG-dUTP incorporation signal, which indicates the extent of unstable DNA ends, we found that Tax induces larger signals than those in control cells. However, in xrs-6 cells deficient for Ku80, this Tax effect was not seen.

Conclusions

The data here demonstrate that clastogenic DNA damage in Tax expressing cells is explained by Tax targeting of Ku80, but not PKcs, XRCC4 or H2AX, which are all proteins directly or indirectly related to the non-homologous end-joining (NHEJ) repair system. Of note, the Ku80 protein plays an important role at the initial stage of the NHEJ repair system, protecting and stabilizing DNA-breaks. Accordingly, HTLV-1 Tax is shown to interfere with a normal cellular protective mechanism for stabilizing DNA breaks. These DNA breaks, unprotected by Ku80, are unstable and are subject to erosion or end-to-end fusion, ultimately leading to additional chromosomal aberrations.