DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases.

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

Strategies for Targeting Retroviral Integration for Safer Gene Therapy: Advances and Challenges.

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for both close proximity to the target DNA and specificity of targeting. Each retrovirus appears to have distinct preferences for DNA sequence and chromatin features at the integration site. Tethering factors determine the preference for chromatin features, but do not affect the subtle sequence preference at the integration site. The sequence preference is likely intrinsic to the integrase protein. New developments may uncouple the requirement for a tethering factor and increase the ability to redirect retroviral integration.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency.

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.

CRISPR Genome Editing Applied to the Pathogenic Retrovirus HTLV-1.

CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4+ T lymphocytes that survive for the lifetime of the host. The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-infected cell growth/survival and prevent immune modulatory effects and ultimately HTLV-1-associated disease. In addition, the HTLV-1 viral genome is highly conserved with remarkable sequence homogeneity, both within the same host and even among different HTLV isolates. This offers more focused guide RNA targeting. In addition, there are several well-established animal models for studying HTLV-1 infection in vivo as well as cell immortalization in vitro. Therefore, studies with HTLV-1 may provide a better basis to assess and advance in vivo genome editing against retroviral infections.

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Nucleosome DNA unwrapping does not affect prototype foamy virus integration efficiency or site selection.

Eukaryotic DNA binding proteins must access genomic DNA that is packaged into chromatin in vivo. During a productive infection, retroviral integrases (IN) must similarly interact with chromatin to integrate the viral cDNA genome. Here we examine the role of nucleosome DNA unwrapping in the retroviral integrase search for a target site. These studies utilized PFV intasomes that are comprised of a tetramer of PFV IN with two oligomers mimicking the viral cDNA ends. Modified recombinant human histones were used to generate nucleosomes with increased unwrapping rates at different DNA regions. These modifications included the acetylmimetic H3(K56Q) and the chemically engineered H4(K77ac, K79ac). While transcription factors and DNA damage sensors may search nucleosome bound DNA during transient unwrapping, PFV intasome mediated integration appears to be unaffected by increased nucleosome unwrapping. These studies suggest PFV intasomes do not utilize nucleosome unwrapping to search nucleosome targets.

Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid.

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging.

Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.

Assembly and Purification of Prototype Foamy Virus Intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

CRISPR/Cas9 Genome Editing to Disable the Latent HIV-1 Provirus.

HIV-1 infection can be successfully controlled with anti-retroviral therapy (ART), but is not cured. A reservoir of cells harboring transcriptionally silent integrated provirus is able to reestablish replicating infection if ART is stopped. Latently HIV-1 infected cells are rare, but may persist for decades. Several novel strategies have been proposed to reduce the latent reservoir, including DNA sequence targeted CRISPR/Cas9 genome editing of the HIV-1 provirus. A significant challenge to genome editing is the sequence diversity of HIV-1 quasispecies present in patients. The high level of quasispecies diversity will require targeting of multiple sites in the viral genome and personalized engineering of a CRISPR/Cas9 regimen. The challenges of CRISPR/Cas9 delivery to the rare latently infected cells and quasispecies sequence diversity suggest that effective genome editing of every provirus is unlikely. However, recent evidence from post-treatment controllers, patients with controlled HIV-1 viral burden following interruption of ART, suggests a correlation between a reduced number of intact proviral sequences and control of the virus. The possibility of reducing the intact proviral sequences in patients by a genome editing technology remains intriguing, but requires significant advances in delivery to infected cells and identification of effective target sites.

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.

Retroviral infection requires integration of the viral genome into the host genome. Recombinant integrase proteins may be purified following bacterial expression. A bulk biochemical assay of integrase function relies on the conversion of supercoiled plasmids to linear or relaxed circles. Single molecule molecular tweezer assays of integrase also evaluate the conversion of supercoiled DNA to nicked and broken species. A bacterial nuclease that co-purifies with retroviral integrase may affect the quantitation of integration activity in bulk or single molecule assays. During purification of retroviral integrase from bacteria, fractions may be screened for contaminating nuclease activity. In order to efficiently separate the nuclease from integrase, the binding affinities of each protein must differ. We find that a co-purifying nuclease may be efficiently separated from integrase based on heparin affinity, but not ionic affinity.

Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration.

Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polß). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polß null cell lines complemented with active site point mutants of Polß. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polß DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

The base excision repair pathway is required for efficient lentivirus integration.

An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.

XPB mediated retroviral cDNA degradation coincides with entry to the nucleus.

Retroviruses must integrate their cDNA to a host chromosome, but a significant fraction of retroviral cDNA is degraded before integration. XPB and XPD are part of the TFIIH complex which mediates basal transcription and DNA nucleotide excision repair. Retroviral infection increases when XPB or XPD are mutant. Here we show that inhibition of mRNA or protein synthesis does not affect HIV cDNA accumulation suggesting that TFIIH transcription activity is not required for degradation. Other host factors implicated in the stability of cDNA are not components of the XPB and XPD degradation pathway. Although an increase of retroviral cDNA in XPB or XPD mutant cells correlates with an increase of integrated provirus, the integration efficiency of pre-integration complexes is unaffected. Finally, HIV and MMLV cDNA degradation appears to coincide with nuclear import. These results suggest that TFIIH mediated cDNA degradation is a nuclear host defense against retroviral infection.

Real-time quantitative PCR and fast QPCR have similar sensitivity and accuracy with HIV cDNA late reverse transcripts and 2-LTR circles.

Real-time fluorescent quantitative PCR (universal QPCR) methods are used routinely in both academic and clinical research to measure HIV cDNA. Fast QPCR allows for faster ramping times between cycles and smaller reaction volumes, but may lose sensitivity and accuracy. We demonstrate that primer sets for HIV late reverse transcripts and 2-LTR circles have similar sensitivity and accuracy with either universal or fast QPCR methods. However, both cost and time are reduced with fast QPCR.