RNA-protein interactions govern antiviral specificity and encapsidation of broad spectrum anti-HIV reverse transcriptase aptamers.

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit HIV-1 replication, but little is known about potential aptamer-specific viral resistance. During replication, RT interacts with diverse nucleic acids. Thus, the genetic threshold for eliciting resistance may be high for aptamers that make numerous contacts with RT. To evaluate the impact of RT-aptamer binding specificity on replication, we engineered proviral plasmids encoding diverse RTs within the backbone of HIV-1 strain NL4-3. Viruses inhibited by pseudoknot aptamers were rendered insensitive by a naturally occurring R277K variant, providing the first demonstration of aptamer-specific resistance in cell culture. Naturally occurring, pseudoknot-insensitive viruses were rendered sensitive by the inverse K277R mutation, establishing RT as the genetic locus for aptamer-mediated HIV-1 inhibition. Non-pseudoknot RNA aptamers exhibited broad-spectrum inhibition. Inhibition was observed only when virus was produced in aptamer-expressing cells, indicating that encapsidation is required. HIV-1 suppression magnitude correlated with the number of encapsidated aptamer transcripts per virion, with saturation occurring around 1:1 stoichiometry with packaged RT. Encapsidation specificity suggests that aptamers may encounter dimerized GagPol in the cytosol during viral assembly. This study provides new insights into HIV-1's capacity to escape aptamer-mediated inhibition, the potential utility of broad-spectrum aptamers to overcome resistance, and molecular interactions that occur during viral assembly.

Effect of ribonucleotides embedded in a DNA template on HIV-1 reverse transcription kinetics and fidelity.

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

Claudin association with CD81 defines hepatitis C virus entry.

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus.

BACKGROUND: The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in ß3-ß4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses. METHODS: Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74. RESULTS: Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT. CONCLUSIONS: The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases.

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.

A novel self-deleting retroviral vector carrying an additional sequence recognized by the viral integrase (IN).

During retroviral integration, the viral integrase recognizes the attachment (att) sequence (formed by juxtaposition of two LTRs ends) as the substrate of integration. We have developed a self-deleting Avian Leukosis and Sarcoma Viruses (ALSVs)-based retroviral vector carrying an additional copy of the att sequence, between neo and puro genes. We observed that: (i) the resulting NP3Catt vector was produced at neo and puro titers respectively smaller and higher than that of the parental vector devoid of the att sequence; (ii) 61% of NP3Catt proviruses were flanked by LTRs; most of them were deleted of internal sequences, probably during the reverse transcription step; (iii) 31% of clones were deleted of the whole 5' part of their genome and were flanked, in 5', by the additional att sequence and, in 3', by an LTR. Integration of these last proviruses was often imprecise with respect to the viral ends. At total, 77% of proviruses had lost the packaging signal and were not mobilizable by a replication-competent virus and 92% had lost the selectable gene in a single round of replication. Although still to improve, the att vector could be considered as an interesting new safe retroviral vector for gene transfer experiments.

Stabilization of the U5-leader stem in the HIV-1 RNA genome affects initiation and elongation of reverse transcription.

Reverse transcription of the Human Immunodeficiency Virus type I (HIV-1) RNA genome is primed by a cellular tRNA-lys3 molecule that binds to the primer binding site (PBS). The PBS is predicted to be part of an extended RNA structure, consisting of a small U5-PBS hairpin and a large U5-leader stem. In this study we stabilized the U5-leader stem of HIV-1 to study its role in reverse transcription. We tested in vitro synthesized wild-type and mutant templates in primer annealing, initiation and elongation assays. Stabilization of the stem inhibits the initiation of reverse transcription, but not the annealing of the tRNA primer onto the PBS. These results suggest that stabilization of the stem results in occlusion of a sequence motif that is involved in an additional interaction with the tRNA-lys3 primer and that is needed to trigger the initiation of reverse transcription. The stable structure was also found to affect the elongation of reverse transcription, causing the RT enzyme to pause upon copying 7-8 bases into the extended base paired stem. The stabilizing mutations were also introduced into proviral constructs for replication studies, demonstrating that the mutant viruses have a reduced replication capacity. Analysis of a revertant virus demonstrated that opening of the stabilized U5-leader stem can restore both virus replication and reverse transcription.

Ribonucleotide reductase genes of Bacillus prophages: a refuge to introns and intein coding sequences.

The ribonucleotide reductase gene tandem bnrdE/bnrdF in SPbeta-related prophages of different Bacillus spp. isolates presents different configurations of intervening sequences, comprising one to three of six non-homologous splicing elements. Insertion sites of group I introns and intein DNA are clustered in three relatively short segments encoding functionally important domains of the ribonucleotide reductase. Comparison of the bnrdE homologs reveals mutual exclusion of a group I intron and an intein coding sequence flanking the codon that specifies a conserved cysteine. In vivo splicing was demonstrated for all introns. However, for two of them a part of the mRNA precursor molecules remains unspliced. Intergenic bnrdE-bnrdF regions are unexpectedly long, comprising between 238 and 541 nt. The longest encodes a putative polypeptide related to HNH homing endonucleases.

The folding pathway of the genomic hepatitis delta virus ribozyme is dominated by slow folding of the pseudoknots.

Hepatitis delta virus (HDV) replicates by a double rolling-circle mechanism that requires self-cleavage by closely related genomic and antigenomic versions of a ribozyme. We have previously shown that the uncleaved genomic ribozyme is subject to a variety of alternative (Alt) pairings. Sequence upstream of the ribozyme can regulate self-cleavage activity by formation of an Alt 1 ribozyme-containing structure that severely inhibits self-cleavage, or a P(-1) self-structure that permits rapid self-cleavage. Here, we test three other alternative pairings: Alt P1, Alt 2, and Alt 3. Alt P1 and Alt 3 contain primarily ribozyme-ribozyme interactions, while Alt 2 involves ribozyme-flanking sequence interaction. A number of single and double mutant ribozymes were prepared to increase or decrease the stability of the alternative pairings, and rates of self-cleavage were determined. Results of these experiments were consistent with the existence of the proposed alternative pairings and their ability to inhibit the overall rate of native ribozyme folding. Local misfolds are treated as internal equilibrium constants in a binding polynomial that modulates the intrinsic rate of cleavage. This model of equilibrium effects of misfolds should be general and apply to other ribozymes. All of the alternative pairings sequester a pseudoknot-forming strand. Folding of ribozymes containing Alt P1 and Alt 2 was accelerated by urea as long as the native ribozyme fold was sufficiently stable, while folding of mutants in which both of these alternative pairings had been removed were not stimulated by urea. This behavior suggests that the pseudoknots form by capture of an unfolded or appropriately rearranged alternative pairing by its complementary native strand. Fast-folding mutants were prepared by either weakening alternative pairings or by strengthening native pairings. A kinetic model was developed that accommodates these features and explains the position of the rate-limiting step for the G11C mutant. Implications of these results for structural and dynamic studies of the uncleaved HDV ribozyme are discussed.

Substitution of Asp114 or Arg116 in the fingers domain of moloney murine leukemia virus reverse transcriptase affects interactions with the template-primer resulting in decreased processivity.

Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.

In search of retrotransposons: exploring the potential of the PCR.

A rapid and universal procedure for isolating reverse transcriptase encoding elements from diverse mammalian genomes using PCR is described. We have designed short, degenerate primers to conserved amino acid domains of retroviral reverse transcriptase. These primers amplify a region, predicted to be 342-396 base pairs for most mammalian retroviruses, that spans several conserved domains of reverse transcriptase. The region encoded by the amplified PCR product contains a number of highly conserved amino acids that aid in identification of either degenerate reverse transcriptase or reverse transcriptase from new, undescribed elements. Additionally, these primers allow the amplification of a piece of DNA large enough to be used for phylogenetic analysis. The primers have been used successfully to isolate a region of three related reverse transcriptases from two mammalian taxa. The generality of this approach is discussed.

Gene amplification of human cytidine deaminase proviral cDNA and increased levels of its mRNA produces enhanced drug resistance to cytosine arabinoside in retroviral-transduced murine fibroblasts.

Hematopoietic toxicity is one of the major problems that limits the effectiveness of many antineoplastic drugs. One approach to overcome this problem is to confer chemoresistance to the hematopoietic cells by gene transfer of drug resistance genes. Human cytidine deaminase (CD) inactivates the cytosine nucleoside analogues, such as cytosine arabinoside (ARA-C), by deamination. We have reported previously that retroviral-mediated gene transfer of CD conferred drug resistance to ARA-C in murine cells. One of the major problems in the use of these vectors is to obtain adequate and prolonged expression of the transferred gene to produce a therapeutic effect in the transduced cells. The objective of this investigation was to determine if it is possible to increase the expression of CD proviral DNA in transduced murine fibroblast cells. We observed that by the use of continuous exposure to increasing concentrations of ARA-C it was possible to enhance drug resistance in the transduced cells. This drug resistance was found to be associated with increases in CD enzyme activity and CD proviral mRNA and by amplification of the proviral CD gene.

PCR and reverse dot hybridization for the detection of endogenous retroviral transcripts.

Two degenerated oligonucleotide primers, known to amplify a fragment of the pol gene in all retroviruses tested so far have been used to amplify pol related sequences from human genomic DNA. Cloning and sequencing these fragments confirm a retroviral relationship for most of them and define 96 groups on the basis of their internal similarity. 96 pol fragments were probed with PCR amplified cDNA in reverse dot hybridization to investigate pol related transcripts. PCR amplified genomic DNA served as a control for contamination of genomic DNA in the RNA preparations. Isopycnic centrifugation in cesium trifluoroacetate yielded RNA with the lowest possible amounts of contaminating DNA. This technique is a powerful and a well-controlled tool for the detection of endogenous retroviral transcripts and may be helpful for investigating the involvement of endogenous retroviruses in various diseases.

An HIV-1 protease screening assay using a non-infectious proviral clone.

An in-vitro assay was developed to screen for HIV-1 protease inhibitors using a non-infectious proviral clone (X19) with a deletion in the envelope gene (Ratner et al., 1991). The proviral clone, X19, was expressed transiently in the COS 7 cell line. The virus was able to replicate as assessed by the presence of p24 in the supernatants, yet the virions produced did not infect CD4 positive cells. To determine the effect of a known protease inhibitor on p24 antigen production, PD 148310 (a Ro 31-8959 analog) was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 h and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. PD 148310 inhibited p24 release in a dose-dependent manner with an IC50 of 23.6 nM. Western blot analysis of the supernatants using a mouse monoclonal antibody against p24 confirmed the presence of a well-defined p24 band in the control lane. At 1000 nM of PD 148310 the p24 band was not detectable, leaving only the unprocessed p55 Gag precursor. This technique is a useful tool to screen for potential HIV-1 protease inhibitors.

Retroviral DNA Transposition: Themes and Variations.

Retroviruses and LTR retrotransposons are transposable elements that encapsidate the RNAs that are intermediates in the transposition of DNA copies of their genomes (proviruses), from one cell (or one locus) to another. Mechanistic similarities in DNA transposase enzymes and retroviral/retrotransposon integrases underscore the close evolutionary relationship among these elements. The retroviruses are very ancient infectious agents, presumed to have evolved from Ty3/Gypsy LTR retrotransposons (1), and DNA copies of their sequences can be found embedded in the genomes of most, if not all, members of the tree of life. All retroviruses share a specific gene arrangement and similar replication strategies. However, given their ancestries and occupation of diverse evolutionary niches, it should not be surprising that unique sequences have been acquired in some retroviral genomes and that the details of the mechanism by which their transposition is accomplished can vary. While every step in the retrovirus lifecycle is, in some sense, relevant to transposition, this Chapter focuses mainly on the early phase of retroviral replication, during which viral DNA is synthesized and integrated into its host genome. Some of the initial studies that set the stage for current understanding are highlighted, as well as more recent findings obtained through use of an ever-expanding technological toolbox including genomics, proteomics, and siRNA screening. Persistence in the area of structural biology has provided new insight into conserved mechanisms as well as variations in detail among retroviruses, which can also be instructive.

Induction of chromosomally integrated HIV-1 LTR requires RBF-2 (USF/TFII-I) and Ras/MAPK signaling.

The HIV-1 LTR is regulated by multiple signaling pathways responsive to T cell activation. In this study, we have examined the contribution of the MAPK, calcineurin-NFAT and TNFalpha-NF-kappaB pathways on induction of chromosomally integrated HIV-1 LTR reporter genes. We find that induction by T-cell receptor (CD3) cross-linking and PMA is completely dependent upon a binding site for RBF-2 (USF1/2-TFII-I), known as RBEIII at -120. The MAPK pathway is essential for induction of the wild type LTR by these treatments, as the MEK inhibitors PD98059 and U0126 block induction by both PMA treatment and CD3 cross-linking. Stimulation of cells with ionomycin on its own has no effect on the integrated LTR, indicating that calcineurin-NFAT is incapable of causing induction in the absence of additional signals, but stimulation with both PMA and ionomycin produces a synergistic response. In contrast, stimulation of NF-kappaB by treatment with TNFalpha causes induction of both the wild type and RBEIII mutant LTRs, an effect that is independent of MAPK signaling. USF1, USF2 and TFII-I from unstimulated cells are capable of binding RBEIII in vitro, and furthermore can be observed on the LTR in vivo by chromatin imunoprecipitation from untreated cells. DNA binding activity of USF1/2 is marginally stimulated by PMA/ ionomycin treatment, and all three factors appear to remain associated with the LTR throughout the course of induction. These results implicate major roles for the MAPK pathway and RBF-2 (USF1/2-TFII-I) in coordinating events necessary for transition of latent integrated HIV-1 to active transcription in response to T cell signaling.

A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes.

Human Topoisomerase II is present in two isoforms, 170KDa alpha and 180KDa beta. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II beta over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-1(93IN101) in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II alpha and beta isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication.