Deciphering the code for retroviral integration target site selection

Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses.     

Human endogenous retroviruses in the primate lineage and their influence on host genomes

Primates emerged about 60 million years ago. Since that time various primate-targeting retroviruses have integrated in the germ line of primate species, and some drifted to fixation. After germ line fixation, continued activity of proviruses resulted in intragenomic spread of so-called endogenous retroviruses (ERVs). Variant ERVs emerged, amplified in the genome and profoundly altered genome structures and potentially functionality. Importantly, ERVs are genome modifiers of exogenous origin. The human genome contains about 8% of sequences of retroviral origin. The human ERVs (HERVs) comprise many distinct families that amplified to copy numbers of up to several thousand. We review here the evolution of several well-characterized HERV families in the human lineage since initial germ line fixation. It is apparent that endogenous retroviruses profoundly affected the genomes of species in the evolutionary lineage leading to Homo sapiens.     

Human endogenous retrovirus family HERV-K(HML-5): status, evolution, and reconstruction of an ancient betaretrovirus in the human genome

The human genome harbors numerous distinct families of so-called human endogenous retroviruses (HERV) which are remnants of exogenous retroviruses that entered the germ line millions of years ago. We describe here the hitherto little-characterized betaretrovirus HERV-K(HML-5) family (named HERVK22 in Repbase) in greater detail. Out of 139 proviruses, only a few loci represent full-length proviruses, and many lack gag protease and/or env gene regions. We generated a consensus sequence from multiple alignment of 62 HML-5 loci that displays open reading frames for the four major retroviral proteins. Four HML-5 long terminal repeat (LTR) subfamilies were identified that are associated with monophyletic proviral bodies, implying different evolution of HML-5 LTRs and genes. Sequence analysis indicated that the proviruses formed approximately 55 million years ago. Accordingly, HML-5 proviral sequences were detected in Old World and New World primates but not in prosimians. No recent activity is associated with this HERV family. We also conclude that the HML-5 consensus sequence primer binding site is identical to methionine tRNA. Therefore, the family should be designated HERV-M. Our study provides important insights into the structure and evolution of the oldest betaretrovirus in the primate genome known to date.     

A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5-7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (approximately 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.     

Genome-Wide Characterization of Endogenous Retroviruses in the Bat Myotis lucifugus Reveals Recent and Diverse Infections

Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ∼25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.     

Hypermutation of an ancient human retrovirus by APOBEC3G

Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome, but all are remnants of ancient retroviral infections and harbor inactivating mutations that render them replication defective. Nevertheless, as viral “fossils,” HERVs may provide insights into ancient retrovirus-host interactions and their evolution. Indeed, one endogenous retrovirus [HERV-K(HML-2)], which has replicated in humans for the past few million years but is now thought to be extinct, was recently reconstituted in a functional form, and infection assays based on it have been established. Here, we show that several human APOBEC3 proteins are intrinsically capable of mutating and inhibiting infection by HERV-K(HML-2) in cell culture. We also present striking evidence that two HERV-K(HML-2) proviruses that are fixed in the modern human genome (HERV-K60 and HERV-KI) were subjected to hypermutation by a cytidine deaminase. Inspection of the spectrum of mutations that are found in HERV-K proviruses in the human genome and HERV-K DNA generated during in vitro replication in the presence of each of the human APOBEC3 proteins unequivocally identifies APOBEC3G as the cytidine deaminase responsible for hypermutation of HERV-K60 and HERV-KI. This is a rare example of the antiretroviral effects of APOBEC3G in the setting of natural human infection, whose consequences have been fossilized in human DNA, and a striking example of inactivation of ancient retroviruses in humans through enzymatic cytidine deamination.     

Retroviral infection of hES cells produces random-like integration patterns

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study I showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.     

Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans.

BACKGROUND: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed. RESULTS: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag-Pro-Pol or Env were each present in multiple proviruses. CONCLUSIONS: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.     

A rare event of insertion polymorphism of a HERV-K LTR in the human genome

Human endogenous retroviruses (HERVs), which constitute a significant part of the human genome, might have a serious impact on primate evolution. Over a hundred insertions of HERV-K(HML-2) family members distinguish the human genome from other primate genomes. However, only three cases of insertion polymorphisms have been reported so far, all for endogenous HERV-K proviruses. This suggests that some retroviral integrations occurred rather recently in human genome evolution. In this report, we describe a very rare case of true insertion polymorphism of a solitary HERV-K LTR in the human genome. Distribution of the LTR-containing allele was tested in 5 Africans and 83 individuals from three Russian populations. The allele frequency appeared to be relatively high in populations of both European and Asian origin. The detected polymorphic LTR could be a useful molecular genetic marker of the corresponding genomic region.     

The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome.

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.     

Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

Background

Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.

Results

To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.

Conclusions

The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome.     

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.     

Common sites of retroviral integration in mouse hematopoietic tumors identified by high-throughput,single nucleotide polymorphism-based mapping and bacterial artificial chromosome hybridization

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near Notch1, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.     

Interplay between endogenous and exogenous human retroviruses

In humans, retroviruses thrive more as symbionts than as parasites. Apart from the only two modern exogenous human retroviruses (human T-cell lymphotropic and immunodeficiency viruses; HTLV and HIV, respectively), ~8% of the human genome is occupied by ancient retroviral DNA [human endogenous retroviruses (HERVs)]. Here, we review the recent discoveries about the interactions between the two groups, the impact of infection by exogenous retroviruses on the expression of HERVs, the effect of HERVs on the pathogenicity of HIV and HTLV and on the severity of the diseases caused by them, and the antiviral protection that HERVs can allegedly provide to the host. Tracing the crosstalk between contemporary retroviruses and their endogenized ancestors will provide better understanding of the retroviral world.     

Interactions between exogenous and endogenous retroviruses

Retroviruses are distinguished from other viruses by several features. Notably, some retroviruses are present as normal elements in the genomes of virtually all vertebrates (endogenous proviruses). Others are exogenous, i.e. horizontally transmitted agents, many of which cause fatal diseases. The endogenous retroviruses are genetically transmitted and to a large extent their significance is uncertain. However, there is evidence suggesting that they contribute to the development of diseases in several animal species. Most importantly, some endogenous retroviruses are capable of interacting with exogenous counterparts through a variety of different mechanisms with serious consequences to the host. Conversely, others are advantageous in that they protect against exogenous retroviruses. In this review various types of interactions between endogenous and exogenous retroviruses are discussed, including receptor interference, recombination, phenotypic mixing, immunological interactions and heterologoustrans-activation.