Key determinants of target DNA recognition by retroviral intasomes

BACKGROUND: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4–6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD). RESULTS: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literature or generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RY dinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated with TSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integration into nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization. Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A), which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloney murine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts with and is catalytically stimulated by cellular bromodomain containing 4 protein. CONCLUSIONS: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bonds into two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bend DNA as sharply as viruses that cleave with 4 bp or 5 bp staggers. For PFV and HIV-1, the selection of signature bases and central flexibility at sites of integration is largely independent of chromatin structure. Furthermore, global Rev-A integration is likely directed to chromatin features by bromodomain and extraterminal domain proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-015-0167-3) contains supplementary material, which is available to authorized users

Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance

APOBEC3 proteins are important for antiretroviral defense in mammals. The activity of these factors has been well characterized in vitro, identifying cytidine deamination as an active source of viral restriction leading to hypermutation of viral DNA synthesized during reverse transcription. These mutations can result in viral lethality via disruption of critical genes, but in some cases is insufficient to completely obstruct viral replication. This sublethal level of mutagenesis could aid in viral evolution. A cytidine deaminase-independent mechanism of restriction has also been identified, as catalytically inactive proteins are still able to inhibit infection in vitro. Murine retroviruses do not exhibit characteristics of hypermutation by mouse APOBEC3 in vivo. However, human APOBEC3G protein expressed in transgenic mice maintains antiviral restriction and actively deaminates viral genomes. The mechanism by which endogenous APOBEC3 proteins function is unclear. The mouse provides a system amenable to studying the interaction of APOBEC3 and retroviral targets in vivo. Virions packaging endogenous protein were isolated from mice for analysis of APOBEC3 without a need for protein overexpression. Biochemical and molecular studies are possible using endogenous protein and viral nucleic acids. Additionally, the effect of APOBEC3-mediated viral mutagenesis and subsequent drug resistance can be modeled in this system. Human APOBEC3G transgenic mice infected with murine retroviruses and treated with an antiretroviral drug allows examination of natural levels of viral replication, APOBEC3 induced hypermutation, and potential viral escape. Studies described herein explore mechanisms of APOBEC3-mediated restriction and drug resistance in vivo. We show that endogenous APOBEC3 protein is efficiently packaged into viral cores, and this protein maintains catalytic activity against artificial substrates. We recovered low levels of G-to-A mutations from natural reverse transcription products, although approximately five to ten fold lower than that thought to be necessary for efficient viral restriction. We show that inhibition of reverse transcription is the main mechanism of restriction in vivo, and can be targeted through virion-packaged or cell-associated protein. Transgenically-expressed human APOBEC3G is instead able to heavily deaminate viral DNA, although frequently to sublethal levels. We assessed the effect of both murine APOBEC3 and APOBEC3G on viral replication in the presence and absence of an antiretroviral drug, and examined viruses for drug resistance mutations. APOBEC3G has a clear effect on the rate of viral mutagenesis in vivo, with the potential to induce drug resistance mutations

Porcine endogenous retroviruses PERV A and A/C recombinant are insensitive to a range of divergent mammalian TRIM5 proteins including human TRIM5

The potential risk of cross-species transmission of porcine endogenous retroviruses (PERV) to humans has slowed the development of xenotransplantation, using pigs as organ donors. Here, we show that PERVs are insensitive to restriction by divergent TRIM5{alpha} molecules despite the fact that they strongly restrict a variety of divergent lentiviruses. We also show that the human PERV A/C recombinant clone 14/220 reverse transcribes with increased efficiency in human cells, leading to significantly higher infectivity. We conclude that xenotransplantation studies should consider the danger of highly infectious TRIM5{alpha}-insensitive human-tropic PERV recombinants

Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity

Basic Research and Clinical Examination of Tumor Virus

Tumor viruses cause cancer; thus, extensive studies are being conducted on them. In this article, we will review the basic medical research on the current clinical genetic tests for tumor viruses such as human papilloma virus, hepatitis B virus, and T-cell leukemia virus. Recently, clinical genetic tests have been developed for quick diagnosis of the tumor virus infection. Additionally, we will review the mutagenesis of murine leukemia retrovirus. In particular, we will focus on the insertional mutagenesis. This will help in deciding the direction of future virus research by combining clinical and basic research

Molecular and phylogenetic analyses of a new Amphotropic murine leukemia virus (MuLV-1313)

BACKGROUND: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice. RESULTS: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains. CONCLUSION: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus

Efficient transduction of LEDGF/p75 mutant cells by complementary gain-of-function HIV-1 integrase mutant viruses

Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase (IN)–binding domain from the lentiviral integration host cofactor lens epithelium–derived growth factor (LEDGF)/p75 are a promising retargeting strategy. We previously proposed the utility of IN mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in human immunodeficiency virus type 1 IN and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in IN, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in IN negatively affected reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ∼75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to IN mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge IN mutant viral and LEDGF/p75 host proteins

Retroviral Integration Site Selection

The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded integrase enzyme, has important consequences for both the virus and the host cell. The analysis of retroviral integration site distribution was facilitated by the availability of the human genome sequence, revealing the non-random feature of integration site selection and identifying different favored and disfavored genomic locations for individual retroviruses. This review will summarize the current knowledge about retroviral differences in their integration site preferences as well as the mechanisms involved in this process

Reconstitution and Characterization of Human Endogenous Retrovirus-K

Retroviruses are a family of clinically significant and scientifically fascinating viruses that infect a wide array of organisms from all vertebrate classes. The two hallmark events in the life cycle of retroviruses are the reverse transcription of the single stranded RNA (ssRNA) genome generating a double stranded DNA (dsDNA) and the integration of this dsDNA into the host genome. Because integration is irreversible and the infected cells are usually difficult to target for elimination in the host, the infection is generally permanent. HIV-1, the most important and well-studied member of all retroviruses, is the causative agent of acquired immune deficiency syndrome (AIDS) for which no vaccine or cure is known. Since recognition of the AIDS epidemic, around 25 million people have died from HIV-1 related causes, including 2 million in 2007. Currently, 33 million people are believed to be living with the virus, with most of these people living in sub-Saharan Africa, where 67% of all infected people reside and 75% of AIDS deaths occurred in 2007. When retroviruses infect germ cells or germ cell progenitors, the virus can become endogenized. These viruses, called endogenous retroviruses (ERV), make up more than 8% of the human genome. The integrated virus will be present in the genome of all cells of the individual derived from the infected germ cell, and be passed on to progeny in a Mendelian manner to following generations. Both chance and the insertion’s effect on the fitness of the host can determine the allelic frequency in the population. Hence, elements which produce large quantities of viral proteins and progeny or elements that insert into a necessary gene will likely reduce the fitness of the host and as an allele will be negatively selected in the host population. Currently, there is no known replication competent HERV, as most proviruses are filled with deletions and premature stop codons. However, one family of Class II HERV, HERV-K(HML-2), seems to have been replicating until recently. The HERV-K(HML-2) family includes human specific members and elements that are polymorphic in the human population, suggesting replication since the divergence of humans from chimpanzees 6 million years ago and potentially more recently as well. In this body of work, the problem of the lack of a replication competent virus sequence is circumvented by deducing a consensus sequence from the youngest set of HERV proviruses. Named HERV-KCON, we find that many of its components are functional individually and together enable infection of target cells in a single-cycle infection system. Using this system, we have characterized the previously unknown aspects of HERV-K(HML-2) life cycle, such as location of assembly and budding, dependency on cell replication, and more extensively, its ntegration site preference. HERV-KCON’s interaction with current anti-retroviral host proteins is accessed, and evidence of the same interaction occurring in vivo is presented in the context of APOBEC3G