Scott D. Gitlin

Human Endogenous Retrovirus K (HML-2) Elements in the Plasma of People with Lymphoma and Breast Cancer

ABSTRACT Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from human endogenous retrovirus K (HERV-K) (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either reverse transcriptase PCR or nucleic acid sequence-based amplification. Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly processed HERV-K (HML-2) Gag and envelope proteins. Finally, using immunoelectron microscopy, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.

Protein Profile of Tax-associated Complexes

Infection with human T-cell leukemia virus type 1 (HTLV-1) results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax, a 40-kDa protein, regulates viral and cellular transcription, host signal transduction, the cell cycle, and apoptosis. Tax has been shown to modulate cellular CREB and NFκB pathways; however, to date, its role in binding to various host cellular proteins involved in tumorigenesis has not been fully described. In this study, we describe the Tax-associated proteins and their functions in cells using several approaches. Tax eluted from a sizing column mostly at an apparent molecular mass of 1800 kDa. Following Tax immunoprecipitation, washes with high salt buffer, two-dimensional gel separation, and mass spectrometric analysis, a total of 32 proteins was identified. Many of these proteins belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling. A few of these proteins, including TXBP151, have been shown previously to bind to Tax. The interaction of Tax with small GTPase-cytoskeleton proteins, such as ras GAP1m, Rac1, Cdc42, RhoA, and gelsolin, indicates how Tax may regulate migration, invasion, and adhesion in T-cell cancers. Finally, the physical and functional association of Tax with the chromatin remodeling SWI/SNF complex was assessed using in vitro chromatin remodeling assays, chromatin remodeling factor BRG1 mutant cells, and RNA interference experiments. Collectively, Tax is able to bind and regulate many cellular proteins that regulate transcription and cytoskeletal related pathways, which might explain the pleiotropic effects of Tax leading to T-cell transformation and leukemia in HTLV-1-infected patients.

Characterization of Human Endogenous Retroviral Elements in the Blood of HIV-1-Infected Individuals

ABSTRACT We previously reported finding the RNA of a type K human endogenous retrovirus, HERV-K (HML-2), at high titers in the plasma of HIV-1-infected and cancer patients (R. Contreras-Galindo et al., J. Virol. 82:9329–9236, 2008.). The extent to which the HERV-K (HML-2) proviruses become activated and the nature of their activated viral RNAs remain important questions. Therefore, we amplified and sequenced the full-length RNA of the env gene of the type 1 and 2 HERV-K (HML-2) viruses collected from the plasma of seven HIV-1-infected patients over a period of 1 to 3 years and from five breast cancer patients in order to reconstruct the genetic evolution of these viruses. HERV-K (HML-2) RNA was found in plasma fractions of HIV-1 patients at a density of ∼1.16 g/ml that contained both immature and correctly processed HERV-K (HML-2) proteins and virus-like particles that were recognized by anti-HERV-K (HML-2) antibodies. RNA sequences from novel HERV-K (HML-2) proviruses were discovered, including K111, which is specifically active during HIV-1 infection. Viral RNA arose from complete proviruses and proviruses devoid of a 5′ long terminal repeat, suggesting that the expression of HERV-K (HML-2) RNA in these patients may involve sense and antisense transcription. In HIV-1-infected individuals, the HERV-K (HML-2) viral RNA showed evidence of frequent recombination, accumulation of synonymous rather than nonsynonymous mutations, and conserved N-glycosylation sites, suggesting that some of the HERV-K (HML-2) viral RNAs have undergone reverse transcription and are under purifying selection. In contrast, HERV-K (HML-2) RNA sequences found in the blood of breast cancer patients showed no evidence of recombination and exhibited only sporadic viral mutations. This study suggests that HERV-K (HML-2) is active in HIV-1-infected patients, and the resulting RNA message reveals previously undiscovered HERV-K (HML-2) genomic sequences.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL)

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Acute Cytomegalovirus Infection Complicated by Vascular Thrombosis: A Case Report

We present a case report of a previously healthy adult with cytomegalovirus infection that was complicated by extensive mesenteric arterial and venous thrombosis. To our knowledge, this is the first reported case of this syndrome in an immunocompetent individual who had no predisposing risk factors for thrombosis, and it demonstrates the propensity for cytomegalovirus to be involved in vascular disease.

HIV infection reveals widespread expansion of novel centromeric human endogenous retroviruses

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV-K (HML-2) family is the most recent group of these viruses to have inserted into the genome, and we have detected the activation of HERV-K (HML-2) proviruses in the blood of patients with HIV-1 infection. We report that HIV-1 infection activates expression of a novel HERV-K (HML-2) provirus, termed K111, present in multiple copies in the centromeres of chromosomes throughout the human genome yet not annotated in the most recent human genome assembly. Infection with HIV-1 or stimulation with the HIV-1 Tat protein leads to the activation of K111 proviruses. K111 is present as a single copy in the genome of the chimpanzee, yet K111 is not found in the genomes of other primates. Remarkably, K111 proviruses appear in the genomes of the extinct Neanderthal and Denisovan, while modern humans have at least 100 K111 proviruses spread across the centromeres of 15 chromosomes. Our studies suggest that the progenitor K111 integrated before the Homo-Pan divergence and expanded in copy number during the evolution of hominins, perhaps by recombination. The expansion of K111 provides sequence evidence suggesting that recombination between the centromeres of various chromosomes took place during the evolution of humans. K111 proviruses show significant sequence variations in each individual centromere, which may serve as markers in future efforts to annotate human centromere sequences. Further, this work is an example of the potential to discover previously unknown genomic sequences through the analysis of nucleic acids found in the blood of patients.

Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis.

Genomic Flexibility of Human Endogenous Retrovirus Type K

ABSTRACT Human endogenous retrovirus type K (HERV-K) proviruses are scattered throughout the human genome, but as no infectious HERV-K virus has been detected to date, the mechanism by which these viruses replicated and populated the genome remains unresolved. Here, we provide evidence that, in addition to the RNA genomes that canonical retroviruses package, modern HERV-K viruses can contain reverse-transcribed DNA (RT-DNA) genomes. Indeed, reverse transcription of genomic HERV-K RNA into the DNA form is able to occur in three distinct times and locations: (i) in the virus-producing cell prior to viral release, yielding a DNA-containing extracellular virus particle similar to the spumaviruses; (ii) within the extracellular virus particle itself, transitioning from an RNA-containing particle to a DNA-containing particle; and (iii) after entry of the RNA-containing virus into the target cell, similar to canonical retroviruses, such as murine leukemia virus and HIV. Moreover, using a resuscitated HERV-K virus construct, we show that both viruses with RNA genomes and viruses with DNA genomes are capable of infecting target cells. This high level of genomic flexibility historically could have permitted these viruses to replicate in various host cell environments, potentially assisting in their many integration events and resulting in their high prevalence in the human genome. Moreover, the ability of modern HERV-K viruses to proceed through reverse transcription and package RT-DNA genomes suggests a higher level of replication competency than was previously understood, and it may be relevant in HERV-K-associated human diseases. IMPORTANCE Retroviral elements comprise at least 8% of the human genome. Of all the endogenous retroviruses, HERV-K viruses are the most intact and biologically active. While a modern infectious HERV-K has yet to be found, HERV-K activation has been associated with cancers, autoimmune diseases, and HIV-1 infection. Thus, determining how this virus family became such a prevalent member of our genome and what it is capable of in its current form are of the utmost importance. Here, we provide evidence that HERV-K viruses currently found in the human genome are able to proceed through reverse transcription and historically utilized a life cycle with a surprising degree of genomic flexibility in which both RNA- and DNA-containing viruses were capable of mediating infection.

Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K–Related Sequences

ABSTRACT Human endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied. IMPORTANCE Retroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.

GLI-2 Modulates Retroviral Gene Expression

ABSTRACT GLI proteins are involved in the development of mice, humans, zebrafish, Caenorhabditis elegans, Xenopus, andDrosophila. While these zinc finger-containing proteins bind to TG-rich promoter elements and are known to regulate gene expression in C. elegans and Drosophila, mechanistic understanding of how regulation is mediated through naturally occurring transcriptional promoters is lacking. One isoform of human GLI-2 appears to be identical to a factor previously called Tax helper protein (THP), thus named due to its ability to interact with a TG-rich element in the human T-lymphotropic virus type 1 (HTLV-1) enhancer thought to mediate transcriptional stimulation by the Tax protein of HTLV-1. We now demonstrate that, working through its TG-rich binding site and adjacent elements, GLI-2/THP actually suppresses gene expression driven by the HTLV-1 promoter. GLI-2/THP has no effect on the HTLV-2 promoter, activates expression from the promoters of human immunodeficiency virus types 1 and (HIV-1 and -2), and stimulates HIV-1 replication. Both effective suppression and activation of gene expression and viral replication require the first of the five zinc fingers, which is not necessary for DNA binding, to be intact. Thus, not only can GLI-2/THP either activate or suppress gene expression, depending on the promoter, but the same domain (first zinc finger) mediates both effects. These findings suggest a role for GLI-2 in retroviral gene regulation and shed further light on the mechanisms by which GLI proteins regulate naturally occurring promoters.