Rafael Contreras-Galindo

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.

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.

NGSQC: Cross-platform quality analysis pipeline for deep sequencing data

BackgroundWhile the accuracy and precision of deep sequencing data is significantly better than those obtained by the earlier generation of hybridization-based high throughput technologies, the digital nature of deep sequencing output often leads to unwarranted confidence in their reliability.ResultsThe NGSQC (N ext G eneration S equencing Q uality C ontrol) pipeline provides a set of novel quality control measures for quickly detecting a wide variety of quality issues in deep sequencing data derived from two dimensional surfaces, regardless of the assay technology used. It also enables researchers to determine whether sequencing data related to their most interesting biological discoveries are caused by sequencing quality issues.ConclusionsNext generation sequencing platforms have their own share of quality issues and there can be significant lab-to-lab, batch-to-batch and even within chip/slide variations. NGSQC can help to ensure that biological conclusions, in particular those based on relatively rare sequence alterations, are not caused by low quality sequencing.

Expression of Human Endogenous Retrovirus Type K (HML-2) Is Activated by the Tat Protein of HIV-1

ABSTRACT Human endogenous retroviruses (HERVs) make up 8% of the human genome. The expression of HERV-K (HML-2), the family of HERVs that most recently entered the genome, is tightly regulated but becomes markedly increased after infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag RNA transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and the expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. This activation was seen especially in lymphocytes and monocytic cells, the natural hosts for HIV-1 infection. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-κB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by chromatin immunoprecipitation assays, mutational analysis of the HERV-K (HML-2) long terminal repeat, and treatments with agents that inhibit NF-κB or NF-AT activation. These studies demonstrate that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection.

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.

Human endogenous retrovirus K gag coassembles with HIV-1 gag and reduces the release efficiency and infectivity of HIV-1

ABSTRACT Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-KCON Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-KCON Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-KCON Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-KCON Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-KCON Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-KCON Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-KCON Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-KCON Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-KCON Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

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.

Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

BackgroundApproximately 8% of the human genome consists of sequences of retroviral origin, a result of ancestral infections of the germ line over millions of years of evolution. The most recent of these infections is attributed to members of the human endogenous retrovirus type-K (HERV-K) (HML-2) family. We recently reported that a previously undetected, large group of HERV-K (HML-2) proviruses, which are descendants of the ancestral K111 infection, are spread throughout human centromeres.ResultsStudying the genomes of certain cell lines and the DNA of healthy individuals that seemingly lack K111, we discover new HERV-K (HML-2) members hidden in pericentromeres of several human chromosomes. All are related through a common ancestor, termed K222, which is a virus that infected the germ line approximately 25 million years ago. K222 exists as a single copy in the genomes of baboons and high order primates, but not New World monkeys, suggesting that progenitor K222 infected the primate germ line after the split between New and Old World monkeys. K222 exists in modern humans at multiple loci spread across the pericentromeres of nine chromosomes, indicating it was amplified during the evolution of modern humans.ConclusionsCopying of K222 may have occurred through recombination of the pericentromeres of different chromosomes during human evolution. Evidence of recombination between K111 and K222 suggests that these retroviral sequences have been templates for frequent cross-over events during the process of centromere recombination in humans.