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Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic., Removal of integrated HIV DNA remains a roadblock for HIV cure. Here, Mancuso et al. show that intravenous administration of an adeno-associated virus-based CRISPR/Cas9 gene editing construct to SIV-infected macaques results in excision of integrated proviral DNA from infected blood cells and tissues known to be viral reservoirs.

We used NOD/SCID mice, also known as NRG, to assess the ability of lentivirus-mediated intravenous delivery of CRISPR in editing the HIV-1 genome from the circulating PBMC engrafts, some of which homed within several animal solid tissues. Lentivirus-mediated delivery of a multiplex of guide RNAs accompanied by Cas9 endonuclease led to the excision of the targeted region of the viral genome positioned within the HIV-1 LTR from the in-vitro-infected human peripheral blood mononuclear cells (PBMCs) embedded in the spleens of NRG mice. Similarly, the treatment of NRG mice harboring PBMC engrafts derived from HIV-1-positive patients with the therapeutic lentivirus eliminated the presence of the viral DNA fragment in the blood, as well as in the spleen, lung, and liver, of the engrafted animals. Sanger sequence analysis of the viral DNA after treatment with the lentiviral vectors expressing Cas9 and gRNAs verified the editing and removal of the proviral DNA fragment from the viral genome at the predicted sites. This proof-of-concept study, for the first time, demonstrates successful excision of the HIV-1 proviral DNA from patient immune cell engrafts in humanized mice upon treatment with lentivirus-expressing CRISPR and causes a decline in the level of replication-competent virus. Keywords: HIV-1, CRISPR/Cas9, gene editing

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barre syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

The emergence of Zika virus in the Americas has followed a pattern that is familiar from earlier epidemics of other viruses, where a new disease is introduced into a human population and then spreads rapidly with important public health consequences. In the case of Zika virus, an accumulating body of recent evidence implicates the virus in the etiology of serious pathologies of the human nervous system, that is, the occurrence of microcephaly in neonates and Guillain-Barre syndrome in adults. Zika virus is an arbovirus (arthropod-borne virus) and a member of the family Flaviviridae, genus Flavivirus. Zika virions are enveloped and icosahedral, and contain a nonsegmented, single-stranded, positive-sense RNA genome, which encodes 3 structural and 7 nonstructural proteins that are expressed as a single polyprotein that undergoes cleavage. Zika genomic RNA replicates in the cytoplasm of infected host cells. Zika virus was first detected in 1947 in the blood of a febrile monkey in Uganda's Zika Forest and in crushed suspensions of the Aedes mosquito, which is one of the vectors for Zika virus. The virus remained obscure, with a few human cases confined to Africa and Asia. There are two lineages of the Zika virus, African and Asian, with the Asian strain causing outbreaks in Micronesia in 2007 and French Polynesia in 2013-2014. From here, the virus spread to Brazil with the first report of autochthonous Zika transmission in the Americas in March 2015. The rapid advance of the virus in the Americas and its likely association with microcephaly and Guillain-Barre syndrome make Zika an urgent public health concern. Ann Neurol 2016;80:479-489.

A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at-risk population is growing larger and includes individuals with HIV-1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements.

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the CNS caused by the human polyomavirus JC (JCV). JCV replication occurs only in human cells and investigation of PML has been severely hampered by the lack of an animal model. The common feature of PML is impairment of the immune system. The key to understanding PML is working out the complex mechanisms that underlie viral entry and replication within the CNS and the immunosurveillance that suppresses the virus or allows it to reactivate. Early models involved the simple inoculation of JCV into animals such as monkeys, hamsters, and mice. More recently, mouse models transgenic for the gene encoding the JCV early protein, T-antigen, a protein thought to be involved in the disruption of myelin seen in PML, have been employed. These animal models resulted in tumorigenesis rather than demyelination. Another approach is to use animal polyomaviruses that are closely related to JCV but able to replicate in the animal such as mouse polyomavirus and SV40. More recently, novel models have been developed that involve the engraftment of human cells into the animal. Here, we review progress that has been made to establish an animal model for PML, the advances and limitations of different models and weigh future prospects.

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.

Many neurological diseases of the central nervous system (CNS) are underpinned by malfunctions of the immune system, including disorders involving opportunistic infections. Progressive multifocal leukoencephalopathy (PML) is a lethal CNS demyelinating disease caused by the human neurotropic polyomavirus JC (JCV) and is found almost exclusively in individuals with immune disruption, including patients with human immunodeficiency virus/acquired immunodeficiency syndrome, patients receiving therapeutic immunomodulatory monoclonal antibodies to treat conditions such as multiple sclerosis, and transplant recipients. Thus, the public health significance of this disease is high, because of the number of individuals constituting the at-risk population. The incidence of PML is very low, whereas seroprevalence for the virus is high, suggesting infection by the virus is very common, and so it is thought that the virus is restrained but it persists in an asymptomatic state that can only occasionally be disrupted to lead to viral reactivation and PML. When JCV actively replicates in oligodendrocytes and astrocytes of the CNS, it produces cytolysis, leading to formation of demyelinated lesions with devastating consequences. Defining the molecular nature of persistence and events leading to reactivation of the virus to cause PML has proved to be elusive. In this review, we examine the current state of knowledge of the JCV life cycle and mechanisms of pathogenesis. We will discuss the normal course of the JCV life cycle including transmission, primary infection, viremia, and establishment of asymptomatic persistence as well as pathogenic events including migration of the virus to the brain, reactivation from persistence, viral infection, and replication in the glial cells of the CNS and escape from immunosurveillance.

Powerful new gene editing techniques promise groundbreaking opportunities for novel therapeutic options to important illnesses, including cancer, genetic disorders [1], and viral infections [2]. These techniques include zinc finger nucleases (ZFN) [3], transcription activator-like effector nucleases (TALEN) [4], and clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) [5, 6]. In particular, CRISPR/Cas9 provides an effective, highly specific, and versatile tool applicable to important human viruses, including HIV-1 [7]. CRISPR/Cas9 is elegant and simple compared to ZFN and TALEN because it uses one or more guide RNAs (gRNA), which are simple to produce and specifically target any sequence in an adaptable and flexible way for different targets, such as viral genes, by changing the gRNA sequence [8]. Cas9 cuts both strands of the DNA target, resulting in a double-strand break (DSB), usually repaired by nonhomologous end-joining (NHEJ), an error-prone arm of the DNA repair pathway that introduces insertions and deletions (InDels) at the break site. While InDel introduction is quite rare, the products of accurate repair are targets for recleavage by CRISPR/Cas9, whereas InDel products are not, and, hence, InDel products accumulate with repeated cleavage cycles [9, 10]. In this short review, we discuss CRISPR/Cas9 in combating human viruses by specifically targeting and disrupting essential viral genes. We will also discuss generation of viral escape mutants resistant to Cas9/gRNA and how this may be overcome.

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.

The human polyomavirus JC (JCV) is the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is extremely common and after primary infection, JCV persists in a latent state. However, PML is a very rare disease suggesting that the virus is tightly regulated. Previously, we showed that NF-κB and C/EBPβ regulate the JCV early and late promoters via a DNA control element, KB, which also mediates the stimulatory effects of proinflammatory cytokines such as TNF-α on JCV gene expression. Other studies have implicated NFAT4 in JCV regulation. We now report that NFAT4 and NF-κB interact at the KB element to co-operatively activate both JCV early and late transcription and viral DNA replication. This interplay is inhibited by C/EBPβ and by agents that block the calcineurin/NFAT signaling pathway. The importance of these events in the regulation of JCV latency and reactivation is discussed.

The fatal CNS demyelinating disease, progressive multifocal leukoencephalopathy (PML), is rare and appears to occur almost always as a consequence of immune dysfunction. Thus, it is associated with HIV/AIDS and also as a side effect of certain immunomodulatory monoclonal antibody therapies. In contrast to the rarity of PML, the etiological agent of the disease, the polyomavirus JC (JCV), is widespread in populations worldwide. In the 40 years since JCV was first isolated, much has been learned about the virus and the disease from laboratory and clinical observations. However, there are many aspects of the viral life cycle and of the pathogenesis of the disease that remain unclear, and our understanding is constantly evolving. In this review, we will discuss our current understanding of the clinical features of PML and molecular characteristics of JCV and of how they relate to each other. Clinical observations can inform molecular studies of the virus, and likewise, molecular findings concerning the life cycle of the virus can guide the development of novel therapeutic strategies.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system that is rare even though the proven etiological agent of PML, the polyomavirus JC (JC virus), is ubiquitous within the human population. The common feature of PML cases appears to be underlying immunosuppression, and PML has gained clinical visibility because of its association with human immunodeficiency virus and AIDS and its occurrence as a side effect of certain immunomodulatory drugs. A hypothesis has gained general acceptance that JC virus causes a primary infection in childhood and enters a latent state, after which immunosuppression allows viral reactivation leading to PML. Nonetheless, many important aspects of PML pathogenesis remain unclear, including the molecular bases of latency and reactivation, the site(s) of latency, the relationship of archetype and prototype virus and the mode of virus transmission within the body and between individuals. In this review, we will revisit these areas and examine what the available evidence suggests.

HIV-1 gene transcription is controlled by the cooperation of viral and host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat (LTR). Previously we showed that the St. John's Wort DING phosphatase, p27SJ, suppresses HIV-1 gene transcription by binding to the viral protein Tat and preventing its nuclear import. Here, we describe the inhibitory effect of p27SJ on the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). This inhibition leads to the suppression of the association of RNAPII with the LTR. Inhibition of binding of RNAPII to LTR by p27SJ resulted in the suppression of LTR transcription elongation and a decrease in LTR transcriptional activity. Another form of the St. John's Wort DING phosphatase, p38SJ, also suppressed binding of RNAPII to the LTR, reduced transcription elongation and was even more powerful than p27SJ in inhibiting the transcriptional activity of the LTR. Our data suggest a possible mechanism by which the p27SJ/p38SJ DING phosphatase can regulate HIV-1 LTR expression by inhibiting phosphorylation of the CTD of RNAPII and suppressing LTR transcription elongation.

BK virus-associated nephropathy (BKVN) occurs in up to 5% of kidney transplants and is a significant cause of graft loss. Four major subtypes of BKV have been described, with the vast majority of individuals persistently infected with BKV Type I (>80% of the population). Sequencing of BKV isolates subcloned from BKVN patients revealed a high percentage of variants in the urine (40%) in the VP1 subtyping region. In vitro analysis of several viral variants revealed that all variants recovered from the urine of BKVN patients produced infectious viral particles and were replication competent in cell culture while some of the variants induced cytopathic changes in infected cells when compared to the major BKV subtype, VP1 Type I. These results suggest that rare BKV VP1 variants are more frequently associated with disease and that some variants could be more cytopathic than others in renal transplant recipients.

Expression of the human polyomavirus JCV genome in several experimental animals induces a variety of neural origin tumors. The viral proteins, T-antigen and Agnoprotein, contribute to the oncogenesis of JCV by associating with several tumor suppressor proteins and dysregulating signaling pathways, which results in uncontrolled cell proliferation. In addition, T-antigen and Agnoprotein have been associated with DNA damage and interfering with DNA repair mechanisms. In this study, we have utilized commercially available tissue arrays of human tumors of various origins and demonstrated the expression of both T-antigen and Agnoprotein in some, but not all, tumors of neural and non-neural origin. Most notably, more than 40% of human glioblastomas and greater than 30% of colon adenocarcinomas express viral proteins. The detection of viral transforming proteins, T-antigen and Agnoprotein in the absence of viral capsid proteins suggests a role for JCV in the development and/or progression of human tumors. These results invite further large-scale investigation on the role of polyomaviruses, particularly JCV in the pathogenesis of human cancer.

The major capsid protein, VP1, of the human Polyomavirus BK (BKV) is structurally divided into five outer loops, referred to as BC, DE, EF, GH, and HI. The BC loop includes a short region, named the BKV subtyping region, spanning nucleotides 1744–1812 and characterized by non-synonymous nucleotide polymorphisms that have been used to classify different strains of BKV into four subtypes. The aim of this study was to determine if the nucleotide changes clustered within the BKV subtyping region may influence the in vitro growth efficiency of the virus. We therefore infected the African Green Monkey kidney cell line Vero with four different viral strains (named BKV I, II, III, and IV) that contained the nucleotide sequences of the BKV subtypes within the same genomic background. Infected cells were followed for 59 days and viral replication was assessed at different time points by Quantitative Real Time PCR (Q-PCR). BKV I, II, and IV were successfully propagated over time in Vero cells, whereas BKV III viral loads progressively decreased during the infection course, demonstrating that the non-synonymous nucleotide polymorphisms of subtype III confer a strong disadvantage for viral replication. Since subtype III differs from all the other subtypes at position 68 of the VP1, where Leu is replaced by Gln, we created viral strains bearing Gln at this position together with the polymorphisms of subtypes I, II, IV and tested their growth in Vero cells. Our results demonstrate that this amino acid substitution does not lower the replication efficiency of subtypes I, II, and IV. In conclusion, this study provides further insights to the importance of the BC loop of BKV in the virus life cycle. In addition, given the effect of the amino acid substitutions of the four BKV subtypes on infectious spread of the virus, our results suggest the need to investigate their potential association with BKV related complications.

The lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins with neurotoxic activity. Among HIV-1 proteins, Vpr has been shown to deregulate expression of various important cytokines and inflammatory proteins in infected and uninfected cells. However, the mechanisms underlying these changes remain unclear. Here, we demonstrate that neurons can take up Vpr that is released into the supernatant of HIV-infected microglia. We also found that administration of recombinant Vpr (rVpr) to human neurons resulted in a slow but sustained elevation of intracellular calcium [Ca(2+)]i. Interestingly, our data also show that [Ca(2+)]i elevation by Vpr leads to ROS production and impairs glutamate signaling in neuronal cells. Vpr disturbs calcium homeostasis through downregulation of endogenous PMCA. Finally, we found that the permeability of the plasma membrane increases in neurons treated with Vpr. Therefore, we conclude that soluble Vpr is a major viral factor that causes a disturbance in neuronal communication leading to neuronal dysfunction. The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will help in the development of new therapeutic approaches.

Activation of viral promoter transcription is a crucial event in the life cycle of several viruses. Hypoxia inducible factor-1 alpha (HIF-1alpha) is an inducible transcription factor whose activity is dependent on environmental conditions, most notably oxygen levels and cellular stress. HIF-1alpha has been implicated in the pathogenesis of several viruses, including HIV-1, HHV-8 and RSV. Under hypoxic conditions or oxidative stress, HIF-1alpha becomes stable and translocates to the nucleus, where it modulates gene transcription. The objective of the present study was to investigate a possible role for HIF-1alpha in the activation of JCV. Glial cell cultures infected with JCV demonstrated a significant increase in the levels of HIF-1alpha, in where it is located to the nucleus. Immunohistochemical studies corroborated upregulation of HIF-1alpha in JCV infected oligodendrocytes and astrocytes in clinical samples of PML compared with normal glial cells from the same samples in which HIF-1alpha expression is weak. CAT assays performed in co-transfected glial cells demonstrated activation of the JCV early promoter in the presence of HIF-1alpha. This activation was potentiated in the presence of Smad3 and Smad4. Finally, chromatin immunoprecipitation assays demonstrated the binding of HIF-1alpha to the JCV control region. These results suggest a role for HIF-1alpha in the activation of JCV; understanding of this pathway may lead to the development of more effective therapies for PML, thus far an incurable disease.

Caspase-3 is the executioner caspase of apoptosis whose activation in mammalian cells represents the last stage of the programmed cell death signaling pathway and the initiation of the lethal digestion of cell proteins. Active caspase-3 is a tetramer composed of two p12 and two p17 subunits derived from cleavage of procaspase-3 during activation. Here, we armed GFP-fusion proteins of both the caspase-3 p12 and p17 subunits with signals from Ig-kappa light chain that allows its efficient secretion from the cells (Sec) and from HIV-1 Tat that facilitates its uptake and nuclear translocation by other cells (NLS). We found that treatment of cells with conditioned media from cells expressing both Sec-GFP-p17-NLS and Sec-GFP-p12-NLS was able to transduce active caspase-3 with consequent cell death of treated cultures. Use of various combinations of constructs demonstrated that both subunits were required and that each one needed to possess both Sec and NLS. Our observations introduce a bidirectional protein transduction system with the ability to introduce active caspase-3 into cells and cause apoptosis. This system may have important therapeutic applications.

Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein–protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82–124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of Sm t-Ag with respect to the regulation of its phosphorylation. Collectively, these results suggest that there is an interplay between agnoprotein, Sm t-Ag and PP2A with respect to the regulation of JCV life cycle and this could be important for the progression of the JCV-induced disease, PML.

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.

To ensure successful replication, HIV-1 has developed a Rev-mediated RNA transport system that promotes the export of unspliced genomic RNA from nuclei to cytoplasm. This process requires the Rev responsive element (RRE) that is positioned in the viral transcript encoding Env protein, as well as in unspliced and singly spliced viral transcripts. We identified Puralpha, a single-stranded nucleic acid binding protein as a cellular partner for Rev that augments the appearance of unspliced viral RNAs in the cytoplasm. A decrease in the level of Puralpha expression by siRNA diminishes the level of Rev-dependent expression of viral RNA. Through its nucleic acid binding domain, Puralpha exhibits the ability to interact with the multimerization and RBD domains of Rev. Similar to Rev, Puralpha associates with RRE and in the presence of Rev forms a complex with slower electrophoretic mobility than those from Rev:RRE and Puralpha:RRE. The interaction of Puralpha with RRE occurs in the cytoplasm where enhanced association of Rev with RRE is observed. Our data indicate that the partnership of Puralpha with Rev is beneficial for Rev-mediated expression of the HIV-1 genome.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the productive infection of oligodendrocytes by the opportunistic polyomavirus JC virus (JCV). Apoptosis is a host defense mechanism to dispose of damaged cells; however, certain viruses have the ability to deregulate apoptotic pathways to complete their life cycles. One such pathway involves survivin, a member of the inhibitor of apoptosis family, which is abundantly expressed during development in proliferating tissues but should be absent in normal, terminally differentiated cells. Immunohistochemistry performed in 20 cases of PML revealed the presence of survivin in JCV-infected oligodendrocytes and bizarre astrocytes within demyelinated plaques. Survivin up-regulation was also found in oligodendroglial and astrocytic cultures infected with JCV. Cell cycle analysis and DNA laddering demonstrated a significantly lower number of cells undergoing apoptosis on JCV infection compared with noninfected cultures; small interfering RNA inhibition of survivin resulted in a dramatic increase in apoptotic cells in JCV-infected cultures. This is the first report describing the activation of survivin by JCV infection in vitro and in PML clinical cases. These observations provide new insights into the anti-apoptotic mechanisms used by JCV to complete its lytic cycle and may suggest new therapeutic targets for PML.

The attention of researchers and clinicians specializing in both multiple sclerosis (MS) and JC virus (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML), was rekindled by the development of PML in two patients with MS enrolled in a clinical trial of combination therapy with natalizumab (Tysabri) and interferon beta-1A (Avonex) in recent years. PML had not been previously reported with either MS or treatment with interferon beta alone. This occurrence of PML with alpha4beta1-integrin inhibition in MS raised a number of issues in terms both of the scientific understanding of these diseases and for the future of immunomodulatory treatment for MS. In this review, we examine the current status of knowledge of the virus, its molecular biology, life cycle, and pathogenetic mechanisms, and how this relates to the basic science and clinical perspectives of MS. A better understanding of the specific steps from JCV infection to the development of PML is key to this issue. Other critical issues for further investigation include the role of alpha4beta1-integrin inhibition by natalizumab in the re-expression of JCV from latent sites and in the inhibition of entry into the brain and peripheral sites.

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the CNS caused by the human polyomavirus JC (JCV). JCV replication occurs only in human cells and investigation of PML has been severely hampered by the lack of an animal model. The common feature of PML is impairment of the immune system. The key to understanding PML is working out the complex mechanisms that underlie viral entry and replication within the CNS and the immunosurveillance that suppresses the virus or allows it to reactivate. Early models involved the simple inoculation of JCV into animals such as monkeys, hamsters, and mice. More recently, mouse models transgenic for the gene encoding the JCV early protein, T-antigen, a protein thought to be involved in the disruption of myelin seen in PML, have been employed. These animal models resulted in tumorigenesis rather than demyelination. Another approach is to use animal polyomaviruses that are closely related to JCV but able to replicate in the animal such as mouse polyomavirus and SV40. More recently, novel models have been developed that involve the engraftment of human cells into the animal. Here, we review progress that has been made to establish an animal model for PML, the advances and limitations of different models and weigh future prospects.

Expression of the viral protein R, Vpr, of HIV-1 affects many biological events in host cells including cell cycle progression, and modulates HIV-1 gene transcription. Earlier studies implicating the cellular protein p21(WAF1) (p21) in regulation of HIV-1 transcription, led us to investigate the functional and physical interaction of Vpr and p21. Our results show that Vpr modestly activated HIV-LTR in cells lacking p21 gene. Here, we describe the mechanisms by which p21 and Vpr leading to stimulation of HIV-1 transcription. Data from the protein-protein interaction experiments revealed the ability of Vpr, p21 and p300 to form a complex. Further, we show that, Vpr interacts with the N- and the C-terminal domains of p21. Furthermore, in cells expressing Vpr, p21 localizes to both the cytoplasm and the nucleus. Interestingly, expression of Vpr alleviates p21-mediated inhibition of cell departure from G1 phase. Expression of a mutant Vpr, with arginine 73 altered to serine, did not affect the ability of p21 to cause cells arrest or its sub-cellular localization. These observations reveal a new cellular partner for Vpr, and provide a new therapeutic avenue for controlling HIV-1 expression.

The retinoblastoma gene product pRb and other members of the Rb family of pocket proteins have a central role in the regulation of cell cycle progression. Soon after its discovery, pRb was found to interact with the transforming oncoproteins of DNA tumor viruses and this led to rapid advances in our understanding of the mechanisms of viral transformation and cell cycle progression. DNA viruses of the polyomavirus family have small, circular, double-stranded DNA genomes contained within non-enveloped icosahedral capsids and are highly tumorigenic in experimental animals. At least three types of polyomavirus infect humans: JC virus (JCV), BK virus (BKV) and Simian Vacuolating virus-40. The early region of these viruses encodes the transforming proteins large T-antigen and small t-antigen, which are involved in viral replication and also promote transformation of cells in culture and oncogenesis in vivo. Binding of T-antigen to pRb promotes the activation of the E2F family of transcription factors, which induce the expression of cellular genes required for S phase. In the context of lytic infection, this cell cycle progression is necessary for viral replication because polyomaviruses rely on S phase-specific host factors for their DNA synthesis. In the context of cellular transformation and tumorigenesis, T-antigen/pRB interaction is an indispensable event.

Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modifications including phosphorylation, which plays a critical role in many aspects of cell function. Previous studies from our and other laboratories indicated that the JC virus (JCV) late regulatory protein, agnoprotein, plays an important role in the JCV life cycle. Agnoprotein contains several potential phosphorylation sites, including Ser7, Ser11, and Thr21, which are potential targets for the serine/threonine-specific protein kinase C (PKC). In this study, we investigated the functional significance of these phosphorylation sites for the activity of agnoprotein. In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. In addition, each of the PKC phosphorylation sites was mutated to Ala singly and in combination, and the effects of these mutations on the JCV life cycle were analyzed. Although the expression of each mutant agnoprotein was detectable during the infection cycle, virus containing each of these mutations failed to propagate. These results contrast with those obtained with an agnoprotein start codon point (Pt) mutant where agnoprotein expression was completely inhibited. The Pt mutant was viable but replicates less efficiently than the wild type (WT). Moreover, conservative substitutions at PKC phosphorylation sites (Ser7, Ser11, and Thr21 to Asp) resulted in a viable virus, which further demonstrate the importance of these sites on agnoprotein function. Further analysis of the mutants by viral release assay and electron microscopy studies revealed that viral particles were efficiently released from infected cells and morphologically indistinguishable from those of WT but were deficient in DNA content. This may account for the defective propagation of the mutants. These results imply that phosphorylated forms of agnoprotein may have essential functions in the viral life cycle and serve as potential targets for therapeutic interventions to limit JCV propagation and JCV-induced diseases.

JC virus (JCV) is the etiological agent of an opportunistic brain infection, progressive multifocal leukoencephalopathy (PML), in AIDS. PML is fatal in approximately 4% of HIV-infected individuals, and although the overall incidence has fallen due to highly aggressive antiretroviral therapy (HAART), this percent has remained steady. It has been shown that the Tat protein of human immunodeficiency virus-1 (HIV-1) interacts in cells with cellular protein Puralpha. This interaction can stimulate transcription of both HIV-1 and JCV genes. HIV-1, however, infects primarily microglia and astrocytes in the brain, whereas JCV infects primarily oligodendrocytes. Although HIV-1 has been shown capable of infecting oligodendrocytes in vitro (Albright et al., 1996), no instance of viral coinfection of such cells with JCV has been reported. Tat is known to be secreted from cells in which it is made. Here we ask whether such exogenous Tat can influence JCV replication in oligodendrocytes. We find that glial cells infected with either HIV-1 or JCV are in proximity at the outer edge of PML lesions. Exogenous Tat is avidly incorporated into cultured KG-1 oligodendroglioma cells over a 72-h period and is colocalized with endogenous Puralpha both nuclear and juxtanuclear. At concentrations in the medium well below the pM range, Tat stimulates several-fold the replication in vivo of DNA initiated at the JCV origin. These results define a pathway by which a protein made by HIV-1 can directly affect the course of infection by another disease-causing virus.

Infectious agents, mainly viruses, are among the few known causes of cancer and contribute to a variety of malignancies worldwide. The agents and cancers considered here are human papillomaviruses (cervical carcinoma); human polyomaviruses (mesotheliomas, brain tumors); Epstein-Barr virus (B-cell lymphoproliferative diseases and nasopharyngeal carcinoma); Kaposi’s Sarcoma Herpesvirus (Kaposi’s Sarcoma and primary effusion lymphomas); hepatitis B and hepatitis C viruses (hepatocellular carcinoma); Human T-cell Leukemia Virus-1 (T-cell leukemias); and helicobacter pylori(gastric carcinoma), which account for up to 20% of malignancies around the globe. The criteria most often used in determining causality are consistency of the association, either epidemiologic or on the molecular level, and oncogenicity of the agent in animal models or cell cultures. However use of these generally applied criteria in deciding on causality is selective, and the criteria may be weighted differently. Whereas for most of the tumor viruses the viral genome persists in an integrated or episomal form with a subset of viral genes expressed in the tumor cells, some agents (HBV, HCV, helicobacter) are not inherently oncogenic, but infection leads to transformation of cells by indirect means. For some malignancies the viral agent appears to serve as a cofactor (Burkitt’s lymphoma-EBV; mesothelioma - SV40). For others the association is inconsistent (Hodgkin’s Disease, gastric carcinomas, breast cancer-EBV) and may either define subsets of these malignancies, or the virus may act to modify phenotype of an established tumor, contributing to tumor progression rather than causing the tumor. In these cases and for the human polyomaviruses the association with malignancy is less consistent or still emerging. In contrast despite the potent oncogenic properties of some strains of human adenovirus in tissue culture and animals the virus has not been linked with any human cancers. Finally it is likely that more agents, most likely viruses, both known and unidentified, have yet to be implicated in human cancer. In the meantime study of tumorigenic infectious agents will continue to illuminate molecular oncogenic processes. © 2004 Elsevier Ltd. All rights reserved.

Polyomavirus nephropathy has become an important complication in kidney transplantation, with a prevalence of 1% to 8%. Unfortunately, the risk factors for polyomavirus nephropathy and renal allograft loss are not well defined. The definitive diagnosis is made through assessment of a kidney transplant biopsy. Recently, noninvasive urine and serum markers have been used to assist in polyomavirus nephropathy diagnosis and monitoring. Primary treatment is immunosuppression reduction, but must be balanced with the risks of rejection. No antiviral treatments for polyomavirus nephropathy have been approved by the Food and Drug Administration. Although cidofovir has shown in vitro activity against murine polyomaviruses, and has been effective in some patients, it is associated with significant nephrotoxicity. Graft loss due to polyomavirus nephropathy should not be a contraindication to retransplantation; however, experience is limited. This review presents potential risk factors, screening, diagnostic and monitoring methods, therapeutic management, and retransplantation experience for polyomavirus nephropathy.

B lymphocytes are known as a potential site for latency and reactivation of the human neurotropic polyomavirus, JC virus (JCV). In light of recent studies on the oncogenicity of JCV and the transforming ability of the JCV early protein, T antigen, we investigated the association of JCV with B-cell lymphomas of the central nervous system. Examination of 27 well-characterized clinical specimens by gene amplification and immunohistochemistry revealed the presence of DNA sequences corresponding to the JCV early genome and the late Agnoprotein in 22 samples and the JCV late genome encoding the viral capsid proteins in 8 samples. Expression of T antigen and that of Agnoprotein by immunohistochemistry were each detected in six specimens. No evidence of the production of viral capsid proteins was observed, ruling out productive infection of JCV in the tumor cells. The results from laser capture microdissection verified the presence of JCV T-antigen sequences in tumor cells with positive immunoreactivity to antibodies against the viral proteins T antigen and Agnoprotein. Due to previous reports demonstrating an association of the Epstein-Barr virus (EBV) with transformation of B lymphocytes, EBV DNA sequences and the EBV transforming protein, latent membrane protein 1 (LMP1), were analyzed in parallel. EBV LMP1 DNA sequences were detected in 16 of 23 samples, and LMP1 expression was detected in 16 samples, 5 of which exhibited positive immunoreactivity to JCV proteins. Double labeling demonstrated coexpression of JCV T antigen and EBV LMP1 in the same cells. The detection of the JCV genome in large numbers of B-cell lymphomas and its coexistence with EBV suggest a potential role for JCV in the pathogenesis of primary CNS lymphoma.

The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.

Polyomaviruses, including JC virus (JCV), BK virus (BKV), and simian virus 40 (SV40) have attracted much attention in the past decade due to their repeated isolation from various human tumors, including those originating from the central nervous system (CNS). JCV and BKV are considered to be ubiquitous human pathogens that become reactivated under impaired physiological conditions such as immunosuppression. Productive replication of JCV and BKV induces diseases such as progressive multifocal leukoencephalopathy in the brain and hemorrhagic or nonhemorrhagic cystitis and nephritis in the kidney. JCV DNA sequences have been isolated from a number of human CNS tumors, including medulloblastoma, ependymoma, and a broad range of glial-origin neoplasms. SV40, once believed to be a monkey virus, has now been isolated from a variety of human cancer cells, including mesothelioma, ependymoma, and non-Hodgkin's lymphoma. In this mini-review, the authors focused their attention on the possible involvement of polyomaviruses, such as JCV, BKV, and SV40, with human brain tumors.

The human polyomavirus, JC virus (JCV), provides an excellent model system to investigate the reciprocal interaction of the immune and nervous systems. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle and upon cytolytic destruction of glial cells, causes the fatal demyelinating disease of the central nervous system (CNS), named progressive multifocal leukoencephalopathy (PML). In this short review, we discuss the molecular pathogenesis of PML by highlighting the role of the immune system in modulating JCV gene activation and replication, and the latency/reactivation of this virus upon immunosuppression. Further, due to the higher incidence of PML among AIDS patients, we further elaborate on the cross-talk between JCV and HIV-1 by direct and indirect pathways that lead to enhanced expression of the JCV genome. © 2002 Wiley-Liss, Inc.

Background: The human neurotropic polyomavirus, JCV, contains an open reading frame within the late region of the viral genome that encodes a 71-amino-acid protein, agnoprotein. Because accumulating evidence supports an association between JCV infection and human brain tumors, including medulloblastomas, we assessed the presence of JCV Agno gene sequences and the expression of agnoprotein in a series of 20 well-characterized medulloblastomas. Methods: Formalin-fixed, paraffin-embedded tumor tissue samples were used for Agno gene amplification and for immunohistochemical analysis. Adjacent sections were stained with an antibody to agnoprotein and with antibodies to cellular structural and regulatory proteins, including the JCV early gene product, T antigen. Results: Analysis of amplified DNA from paraffin-embedded samples revealed the presence of the Agno gene in 11 (69%) of 16 samples. Immunohistochemical analysis showed cytoplasmic localization and widespread distribution of agnoprotein in the neoplastic cells in 11 (55%) of 20 samples. The JCV early gene product, T antigen, was present in the nucleus of some, but not all, of the neoplastic cells. Some medulloblastoma samples that expressed agnoprotein had no sign of T-antigen expression, p53 was detected in only six of the 11 tumors in which agnoprotein was expressed. None of the 20 samples showed expression of the viral late capsid proteins, ruling out productive infection of the tumor cells with JCV. Conclusions: Our data provide evidence that the JCV late gene encoding the auxiliary agnoprotein is expressed in tumor cells. The finding of agnoprotein expression in the absence of T-antigen expression suggests a potential role for agnoprotein in pathways involved in the development of JCV-associated medulloblastomas.

Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients.

SUMMARY About a fifth of all human cancers worldwide are caused by infectious agents. In 12% of cancers, seven different viruses have been causally linked to human oncogenesis: Epstein-Barr virus, hepatitis B virus, human papillomavirus, human T-cell lymphotropic virus, hepatitis C virus, Kaposi's sarcoma herpesvirus, and Merkel cell polyomavirus. Here, we review the many molecular mechanisms of oncogenesis that have been discovered over the decades of study of these viruses. We discuss how viruses can act at different stages in the complex multistep process of carcinogenesis. Early events include their involvement in mutagenic events associated with tumor initiation such as viral integration and insertional mutagenesis as well as viral promotion of DNA damage. Also involved in tumor progression is the dysregulation of cellular processes by viral proteins, and we describe how this has been investigated by studies in cell culture and in experimental animals and by molecular cellular approaches. Also important are the molecular mechanisms whereby viruses interact with the immune system and the immune evasion strategies that have evolved.

JC virus (JCV) causes progressive multifocal leukoencephalopathy, a demyelinating disease in brains of individuals with AIDS. Previous work has shown that the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), can interact with cellular protein Purα to enhance both TAR-dependent HIV-1 transcription and JCV late gene transcription. Tat has been shown to activate JCV transcription through interaction with Purα, which binds to promoter sequence elements near the JCV origin of replication. DNA footprinting has shown that Purα and large T-antigen cooperatively interact at several binding sites in the origin and transcriptional control region. Overexpression of Purα inhibits replication initiated at the JCV origin by T-antigen. In transfected glial cells Tat reversed this inhibition and enhanced DNA replication. In an in vitro replication system maximal activation by Tat, more than sixfold the levels achieved with T-antigen alone, was achieved in the presence of Purα. Effects of mutant Tat proteins on both activation of replication and binding to Purα have revealed that Cys22 exerts a conformational effect that affects both activities. The origin of an archetypal strain of JCV was less susceptible to activation of replication by Tat relative to the rearranged Mad-1 strain. These results have revealed a previously undocumented role for Tat in DNA replication and have indicated a regulatory role for JCV origin auxiliary sequences in replication and activation by Tat.

Results from a large volume of studies have shown that the auxiliary proteins produced by various eukaryotic viruses may play a critical role in orchestrating the viral lytic cycle and the state of viral latency. This group of proteins has a diverse effect on various stages of infection including transcription (11, 13, 14, 27, 41, 61), translation (19), replication (12, 35, 50), viral assembly (39), release of viral particles (51, 56), and export of viral transcripts from nucleus to cytoplasm (15). In addition, this group of viral proteins may have an impact upon host function and, by deregulating expression of key cellular genes, contribute to the pathogenesis of viral-induced disease. The human neurotropic polyomavirus, JC virus (JCV), contains an open reading frame for expressing a 71-amino-acid peptide whose function in the viral life cycle remains unknown. Clinically, replication of JCV in glial cells induces the fatal demyelinating disease of the brain, progressive multifocal leukoencephalopathy (PML) (5, 8, 60). The genome of JCV is composed of a double-stranded covalently linked circular DNA which is composed of three functional regions (17), including viral early and late coding regions and the viral noncoding regulatory region (Fig. ​(Fig.1A).1A). The viral early coding region encodes two regulatory proteins, small and large T antigens. Although little is known about the function of small t antigen, the large T antigen was shown to be a multifunctional phosphoprotein which, by interacting with several cellular proteins including Purα and YB-1 (18, 47, 48), can modulate both the initiation of viral DNA replication and activation of JCV late gene transcription. In addition to small and large T antigens, the viral early genome encodes several spliced variants of early proteins, due to alternative splicing of the early transcripts (57). These variants of T antigen have been shown to differentially interact with the retinoblastoma family of tumor suppressor proteins (7). Additionally, JCV T antigen is oncogenic and its expression can induce development of tumors of neural origin in experimental animals (31, 49, 58), and its genome has been found in several human tumors (32, 33, 45). The viral late coding region is responsible for expression of three structural proteins, VP1, VP2, and VP3, all of which participate in the formation of viral capsids (36). In addition, the leader of the late transcripts contains an open reading frame for the 71-amino-acid Agno protein. FIG. 1 Structural organization of the JCV genome and Agno protein. (A) JCV genomic structure, depicting the direction of the genes encoding the early and late proteins with arrows. Early genes encode small and large T antigens. Late genes encode viral capsid ... JCV is closely related to the other polyomaviruses, including the simian vacuolating virus (SV40) and the human BK virus (BKV) (16, 17). These viruses share significant sequence homology, particularly in their coding regions (16). JCV and SV40 exhibit approximately 80% homology in their early coding regions, close to 70% homology in their late coding sequences for VP1, VP2, and VP3, and less than 70% homology in the region corresponding to Agno protein. The highest degree of divergence between JCV and SV40 Agno proteins is clustered at the far carboxyl terminal regions (Fig. ​(Fig.1B).1B). JCV and BKV also share a similar degree of homology in those respective regions. The regulatory region of JCV is distinct from those of SV40 and BKV and is responsible for cell-type-specific transcription of the viral genome in central nervous system (CNS) cells (1, 28, 42, 43, 54, 55). Earlier studies on both BKV and SV40 established that the Agno gene is expressed during the late phase of infection (25, 26, 30, 38, 46). Mutational analysis of SV40 Agno protein revealed the importance of this protein in the regulation of the SV40 lytic cycle and virion production (4, 38, 40). While the mechanisms involved in this process remain unknown, it is postulated that SV40 Agno protein may have a functional role at the level of viral assembly (26, 37, 38, 40), maturation (24, 26), transcription, and translation (3, 21, 22). In this study, we examined the effect of JCV Agno protein on transcription and replication of the viral genome in glial cells, in the absence and presence of the viral early protein T antigen. We demonstrate that functional and structural interaction of Agno protein with T antigen results in suppression of viral gene expression and DNA replication in glial cells.

JC virus (JCV) is a human polyomavirus known as the causative agent of the fatal demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML). Further, in experimental animals this virus causes a broad range of tumors of central nervous system origin. Recent studies have suggested the association of JCV with several human tumors most notably malignant brain tumors of childhood, medulloblastoma. The development of tumors by JCV is most likely through mechanisms involving inactivation of tumor suppressors and de-regulation of signaling pathways such as Wnt by the viral early protein, T-antigen. The neurotropic nature of JCV along with the overwhelming evidence for its oncogenic potential in laboratory animals and its detection in significant numbers of human medulloblastomas invite the re-evaluation of the role for JCV in the development of human brain tumors.

The lytic phase of JC virus (JCV) appears to be highly complex and remains elusive. A growing body of experimental evidence suggests that the regulation of JCV gene expression and replication requires, in addition to the presence of specific transcription factors, cooperativity between viral and cellular regulatory proteins. This cooperativity may be accomplished by physical interaction of the participant proteins on and/or off the viral DNA sequence. Here, we present evidence of specific physical and functional interaction between a cellular factor, YB-1, and the JCV early protein, T-antigen, and showed that both proteins play important roles in JCV gene transcription. Additionally, our data indicate that YB-1 also functionally interact with another viral protein, designated agnoprotein, which is expressed late during the course of infection, adding further complexity to the currently known picture on JCV gene regulation.

Examination of the primary tumor of glioblastoma multiforme and its recurrence for their association with JC virus revealed that, while the viral genome is present in both initial and recurrent tumors, expression of the viral oncoprotein T-antigen occurs only in the recurrent tumor cells. Accordingly, the level of inducible cellular transcription factors, including the p65 subunit of NF-kappaB and YB-1, which have the ability to stimulate JCV gene expression, was found to be higher in the recurrent tumor cells. These observations suggest that induction of the regulatory factors after resection of the primary tumor may have reactivated JC virus gene expression and led to redevelopment of the tumor in brain.

In recent years, there has been mounting evidence pointing to the association of polyomaviruses with a wide range of human cancers. The human neurotropic polyomavirus, JCV, infecting greater than 75% of the human population produces a regulatory protein named T-antigen which is expressed at the early phase of viral lytic infection and plays a critical role in completion of the viral life cycle. Furthermore, this protein has the ability to transform neural cells in vitro and its expression has been detected in several human neural-origin tumors. To further investigate the oncogenic potential of the JCV early protein in vivo, transgenic mice expressing JCV T-antigen under the control of its own promoter were generated. Nearly 50% of the animals developed large, solid masses within the base of the skull by 1 year of age. Evaluation of the location as well as histological and immunohistochemical data suggest that the tumors arise from the pituitary gland. As T-antigen is known to interact with several cell cycle regulators, the neoplasms were analysed for the presence of the tumor suppressor protein, p53. Immunoprecipitation/Western blot analysis demonstrated overexpression of wild-type, but not mutant p53 within tumor tissue. In addition, co-immunoprecipitation established an interaction between p53 and T-antigen and overexpression of p53 downstream target protein, p21/WAF1. This report describes the analysis of inheritable pituitary adenomas induced by expression of the human polyomavirus, JCV T-antigen in transgenic mice where T-antigen disrupts the p53 pathway by binding to and sequestering wild-type p53. This animal model may serve as a useful tool to further evaluate mechanisms of tumorigenesis by JCV T-antigen.

The 96-amino-acid-long human immunodeficiency virus type 1 virion-encoded accessory protein Vpr is of particular interest, as this protein, which is found in association with viral particles, can exert a regulatory effect on both virus replication and host cell function. Evidently, Vpr, through interaction with several host regulatory proteins, can modulate transcription from the viral long terminal repeat promoter. Expression of Vpr in cells results in deregulation of cell proliferation during the cell cycle pathway at the G 2 stage. Vpr has unique structural features consisting of multiple functional domains. In this study, we have focused on the leucine/isoleucine-rich domain near the carboxyl terminus of Vpr at residue 73 (arginine) and have demonstrated that alterations at this residue result in ablation of transcriptional activity of Vpr and its ability to block cell cycle events at the G 2 stage. Interestingly, substitution mutations at R73 have resulted in a peptide with dominant negative activities on wild-type functions in transcription and host proliferation events. Results from in vitro and in vivo protein-protein interaction studies have revealed that functionally inactive mutant Vpr can be associated with wild-type protein, presumably through the N-terminal regions of the protein which have been shown to be important for Vpr oligomerization. Thus, it is likely that complexation of the mutant Vpr with wild-type protein functionally inactivates Vpr. The importance of these findings in light of the development of therapeutic strategies is discussed.

Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5′ ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal promoter containing TAR sequences, whereas mutant YB-1 with no ability to bind to Tat and TAR failed to affect Tat-mediated activation. Expression of mutant YB-1(1–125), which binds to Tat but not RNA, decreased Tat- mediated enhancement of virus transcription. These observations suggest that while full-length YB-1 may function as a facilitator and, by interaction with both Tat and TAR, increase the level of Tat:TAR association, mutant YB-1 with no TAR binding activity, by complexing with Tat, may prevent Tat interaction with TAR. The importance of these findings in light of the proposed mechanism of Tat function is discussed.