- Open Access
Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration
© Tervo et al; licensee BioMed Central Ltd. 2008
- Received: 16 May 2008
- Accepted: 08 July 2008
- Published: 08 July 2008
The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans.
Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse B1 elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines.
In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.
- Nuclear Import
- Preintegration Complex
- Critical Host Factor
Human immunodeficiency virus type 1 (HIV-1) displays a highly restricted host and cell tropism and is only capable of efficient replication in primary and immortalized T-cells and macrophages of human origin. Cells from native mice do not or only inefficiently support various steps of the HIV-1 replication cycle [1–7]. The precise mapping of some of these species-specific barriers has, on one hand, facilitated the identification and molecular characterization of critical host factors, and, on the other hand, highlighted the complexity of the task to develop genetically altered mice that are fully permissive for HIV-1 infection.
The by far most prominent category of barriers thus far identified in mouse cell lines appears to be recessive in nature. Blocks in this category are characterized by an inability of mouse orthologues of cellular proteins that are essential cofactors for HIV-1 replication in human cells to support distinct replication steps of the virus. HIV-1 entry provides a compelling example since CD4 and the chemokine co-receptor CCR5 from mice bind the HIV-1 envelope glycoprotein with presumably only low affinity and this interaction is insufficient to support virion fusion [4, 5, 8]. Moreover, the discovery that expression of the human HIV-1 receptor complex largely overcomes the entry restriction has provided the rationale for the development of permissive multi-transgenic mouse and rat models through a block-by-block humanization . Along these lines, expression of the human version of the Tat-interacting protein cyclin T1 was shown to boost HIV-1 transcription in mouse cells in vitro and in vivo [3, 7, 10–14]. Additional, less-defined blocks in the late phase of the HIV-1 replication cycle in NIH3T3 cells add up to a profound drop in the yield of viral progeny (up to 104-fold) from a single round of replication [4, 5, 15]. Also these late-stage barriers in mouse fibroblasts display a recessive phenotype and likely result from non-functional mouse cofactors since they can be surmounted in mouse-human heterokaryons [4, 5, 15–17].
Cellular restriction factors, defining a different class of barrier characterized by dominant inhibitory activities, can interfere with lentiviral replication in a species-specific manner. Of potential relevance in the rodent context, the incorporation of the cytidine deaminase APOBEC3G of mouse origin into particles cannot, in contrast to its human orthologue, be counteracted by the HIV-1 Vif protein, resulting in a pronounced reduction in particle infectivity . Providing another example, an early post-entry barrier has been reported for a SIVmac reporter virus in NIH3T3 cells, which displayed typical characteristics of a restriction factor .
However, most of these replication barriers in mice have been described in fibroblast cell lines and the efficiency of different steps of the HIV-1 replication cycle in more relevant target cells has remained elusive. More recently, a severe post-entry defect has been reported in infected mouse T-cells [19–21]. One study mapped this defect to a reduced efficiency of reverse transcription and nuclear import of the HIV-1 pre-integration complex . A second study, in contrast, suggested nuclear import to be the sole cause of the early-phase restriction .
Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans. Starting from a single viral challenge, the efficiency of virus entry, reverse transcription, nuclear import, the frequency of integration, as well as transgene expression off a cytomegalovirus (CMV) immediate early promoter or off the HIV-1NL4-3 LTR were carefully monitored to pinpoint the restriction.
HIV-1-infected mouse T-cell lines do not express a CMV-driven GFP reporter despite efficient virion entry
We first sought to establish a quantitative relationship between the ability of HIV-1 virions to enter T-cells of mouse and human origin and, subsequently, to express a reporter gene in these target cells. To ensure comparable conditions in the cross-species comparisons, we employed an HIV-1 based lentiviral vector encoding for GFP driven by a cytomegalovirus immediate early promoter (HIV-CMV-GFP), which was pseudotyped with the vesicular stomatitis virus glyco-protein (VSV-G). Notably, the expression of GFP from this vector is not influenced by HIV-1 Tat/cyclin T1-dependent, potentially species-specific differences in LTR transactivation . Through incorporation of enzymatically active β-lactamase-Vpr fusion proteins (BlaM-Vpr) during virus production the efficiency of HIV-1 entry into target cells was specifically measured by CCF2 substrate cleavage in a flow cytometry-based virion-fusion assay [22, 23].
HIV-1 reverse transcription and nuclear import occur efficiently in mouse T-cells
To characterize at which step of the replication cycle following entry HIV-1 encounters a block in murine T-cells, levels of late HIV-1 cDNAs and episomal 2-LTR circles were analyzed as markers for reverse transcription and nuclear import of the pre-integration complex, respectively. DNA was extracted from infected mouse and human T-cell lines (from the experiment shown in Figs. 1, 2), aliquots of which were harvested 24 h p.i. and analyzed by real-time PCR. The HIV-1 cDNA species were quantified using established protocols, specificity controls, and quantitative standards for either HIV-1 cDNA species and normalized to cellular DNA levels, which were determined in a parallel reaction by amplification of a cellular gene [6, 25].
Establishment and validation of a quantitative nested PCR to detect integrated HIV-1 DNA in the mouse genome
To establish a standard for quantitative analyses of integration into the mouse genome, a stable polyclonal population of NIH3T3 fibroblasts containing integrated HIV-1 provirus was generated by infection with VSV-G HIV-1NL4-3 GFP at a low MOI, cell passage for 7 weeks to allow complete loss of all unintegrated HIV-1 cDNA species, and subsequent enrichment of GFP-positive cells by flow cytometric sorting (thereafter referred to as NIH3T3Pint cells), in principle as reported previously for the rat species . Since these NIH3T3Pint cells no longer contain unintegrated HIV-1 cDNA species, the absolute number of HIV-1 integrants was accurately quantified by the number of total HIV-1 cDNA copies (54 HIV-1 cDNA copies per ng DNA), thus providing a faithful reference for the integration PCR standard in the mouse genome. Fig. 4B depicts a typical mouse HIV-1 integration standard plotted as a function of the natural logarithm of the concentration of HIV-1 versus the PCR cycle threshold. This standard has a dynamic range of over 3 logs with a highest copy number of 36.741, and both the slope and R2 value were considered as quality controls in individual experiments.
Next, the mouse integration PCR was further validated in a dynamic infection context. Levels of total HIV-1 cDNA and integrants were quantified in parental NIH3T3 fibroblasts infected with either the integration-competent (IN wt) HIV-CMV-GFP or an integration-defective isogenic HIV-1 vector (IN(D64V)), the latter carrying a catalytic core mutation in integrase . One day after infection, high levels of total HIV-1 cDNA were found in NIH3T3 cells challenged with either lentiviral vector, while only background levels could be amplified from efavirenz-treated controls (Fig. 5B). In DNA extracted on day 7 p.i., integrants were readily amplified by the newly developed real-time PCR protocol in NIH3T3 cells infected with the IN wt vector, while the level of provirus formation was severely reduced with the IN(D64V) mutant (Fig. 5C). Collectively, a real-time PCR for the specific detection and absolute quantification of HIV-1 integrants in the genome of infected mouse cells was established and validated.
Levels of HIV-1 integrants are severely reduced in infected mouse T-cell lines
Evidence for cyclin T1-independent transcriptional deficit in certain T-cell lines
To gain insight into the quantitative relationship between vector integrants and transgene expression, the ratio of the percentage of T-cells expressing GFP from the CMV IE promoter and levels of integrants per cell was calculated, for convenience termed Transgene-Expression-per-Integrant. For the residual integrants in mouse R1.1 T-cells (Fig. 6A, B), reporter gene expression was efficient, resulting in a Transgene-Expression-per-Integrant value in a range similar to human MT-4 cells (Fig. 6C). In contrast, the Transgene-Expression-per-Integrant value in infected S1A.TB cells was 34-fold lower than R1.1 (Fig. 6C), indicating that integrants in this mouse T-cell line frequently do not result in a detectable gene expression. This suggests that at least in some T-cell lines a defect in transgene expression from residual integrants may impose an additional peri-integrational limitation in the mouse species.
Following infection with a near-full length HIV-1NL4-3, mouse T-cells do not support early viral gene expression
In an attempt to circumvent this technical limitation, we reasoned that the reduced ability of mouse T-cells to form proviruses should be reflected in a loss of cell-associated HIV-1 cDNA during prolonged passage. Consequently, cells were challenged with the replication-defective VSV-G HIV-1NL4-3GFP carrying BlaM-Vpr and continuously cultivated for four weeks. First, levels of virus entry (6 h p.i.; data not shown), de novo synthesized HIV-1 cDNA (Fig. 8B), and 2-LTR circles (data not shown) were fairly comparable on 1 day p.i., confirming the intact early phase of the replication cycle and successful nuclear import of the preintegration complex. In contrast, a massive loss of cell-associated HIV-1 cDNA was observed in all three mouse T-cell lines at day 14 p.i. (Fig. 8B; 102- to > 2317-fold reduction) with levels in passaged TIMI.4 cells at background. On day 28 p.i., levels of amplified HIV-1 cDNA were further reduced in R1.1 and S1A.TB cells. Collectively, these results provide additional evidence that HIV-1 cDNA, despite nuclear localization, cannot efficiently integrate into the genome of mouse T-cell lines, resulting in a largely abortive infection.
The mapping of HIV-1 replication barriers in non-human cells has allowed the identification and molecular characterization of critical host factors [3, 31] and potent restriction factors [32, 33]. This has, on one hand, expanded our understanding of fundamental processes in the host-virus interaction and, on the other hand, fuelled efforts to develop genetically altered rodents and non-human primates that are highly permissive for HIV-1 [1, 14, 34, 35]. In the current study we demonstrate that T-cells from mice restrict HIV-1 replication at the level of integration.
We examined the fate of the virion and viral genome through consecutive steps in the early infection phase following a single challenge with dual-reporter HIV-1 virions. To circumvent potential HIV-1 Tat/cyclin T1-dependent, species-specific differences in LTR transactivation , we used an HIV-based vector encoding for GFP driven by a CMV promoter. This experimental approach corroborated the finding reported in three earlier studies that primary and immortalized T-cells from mice harbor a severe post-entry restriction for HIV-1, in the context of infections with virions carrying either HIV envelopes or VSV-G [19–21]. Of note, analogous experiments using a near-full length HIV-1NL4-3, in which the GFP reporter is driven off the 5'-LTR (Fig. 7), demonstrated a similar defect in the Relative-Post-Entry-Efficiency in mouse T-cells.
Carefully controlled virion fusion and real-time PCR analyses showed that reverse transcription per fused HIV-1 particle is at least as efficient in T-cells from mice as in their human counterparts. Furthermore, relative levels of 2-LTR circles were comparable or in some instances even markedly elevated in mouse T-cells (Figs. 3D, 8A), demonstrating transfer of the pre-integration complex into the nucleus. In contrast, Baumann et al.  reported reduced efficiencies at both of these replication steps in the identical mouse T-cell lines used in our study. The reason for these discrepancies is currently unclear. In this report  we noted several differences to our study, including (i) a side-by-side comparison of infected mouse T-cells only with mouse NIH3T3 fibroblasts, but not human T-cells, (ii) a lack of correlative evaluations of consecutive replication steps, (iii) a lack of control for residual proviral plasmid contamination in virus inocula, and (iv) quantification of HIV-1 cDNA species without a PCR-based normalization for corresponding cell equivalents.
In line with our observations, Tsurutani et al.  found that reverse transcription proceeds normally in mouse T-cells and that levels of 2-LTR circles were similar in the cross-species comparison following an HIV-1 IN wt infection. Using a cassette ligation-mediated PCR these authors noted a qualitative reduction in integrants in primary lymphocytes from mice. However, based on 2- to 8-fold lower relative 2-LTR circle levels for infections with an integrase-defective HIV-1, Tsurutani et al. located the restriction exclusively at the level of nuclear import in mouse cells. Conceivably, the abundance of 2-LTR circles can be affected by a large number of parameters including the levels of reverse transcribed viral DNA, the efficiency of nuclear import of the pre-integration complex, the efficiency of integration, the activity of cellular ligases of the non-homologous DNA end joining pathway, the formation frequency of other episomal HIV-1 cDNA species (1-LTR circles and auto-integrants), the degradation kinetics of episomes, the time point p.i. as well as the frequency of cell division [36–39]. Of note, the ratio of 2-LTR circles per total HIV-1 cDNA was ~100- to 1000-fold lower in the identical T-cell lines infected with the lentiviral vector (0.008% – 0.01%; Fig. 2A, B) compared with the near full-length HIV-1NL4-3 virus, 0.6%; 1.2% and 25.6% (see Fig. 8A, B). In addition to the difficulties in assessing 2-LTR circle levels discussed above, this also suggests marked construct-dependent differences in the formation of these HIV-1 cDNA episomes. While the presence of 2-LTR circles can be taken as unambiguous proof for nuclear transfer of pre-integration complexes, a quantitative assessment of this episomal DNA species, in light of the plethora of factors influencing its steady-state levels, in our view, does not allow a selective examination and conclusive interpretation of the efficiency of nuclear import in a cross-species comparison.
As a technical advance, we established a nested real-time PCR to specifically detect integrants in the mouse genome. Of note, repetitive B1 elements in the mouse genome are approximately 5-fold less abundant than Alu elements in the human genome (2% versus 10%) , which potentially renders the mouse HIV-1 integration PCR slightly less sensitive. This does, however, not affect the quantification of absolute integrant numbers per genome equivalent, since each species-specific HIV-1 integration standard was calibrated against a total HIV-1 cDNA quantification in long-term passaged NIH3T3int and HeLaint cells. This strategy thus allows a direct comparison of absolute levels of integrants between two species.
First and most importantly, we identify and quantitatively describe a severe limitation at the level of provirus formation in infected mouse T-cell lines, in which pre-integration complexes had entered the nucleus. Based on the above arguments, we cannot formally exclude a quantitative limitation in nuclear import in mouse T-cells based on the 2-LTR circle analyses, however, we can clearly demonstrate a paramount defect at the level of integration. Different degrees of integration impairment were noted with TIMI.4 cells displaying the most drastic restriction, while Relative-Integration-Frequency values in R1.1 and S1A.TB cells were 18- to 54-fold lower compared to human MT-4 cells. As an important characteristic, the barrier was not overcome at high MOIs which favors the hypothesis of a lacking supportive cellular factor rather than the presence of an inhibitory or restriction factor. Furthermore, analysis of the Transgene-Expression-per-Integrant suggests that a cyclin T1-independent transcriptional defect may impose an additional peri-integrational limitation for a productive HIV-1 infection at least in some mouse T-cell lines. Conceptually, integration into transcriptionally unfavorable sites in the host genome [41, 42] could result in such a phenotype.
The fate of HIV-1 cDNA following nuclear entry is a step in the retroviral replication cycle that only recently is being recognized to be dependent on and modulated by specific host factors. Most notably, lense epithelium-derived growth factor (LEDGF/p75) is a chromatin-associated host protein that directly interacts with integrase and apparently tethers the pre-integration complex to the chromosome [43–47]. LEDGF/p75 is, on one hand, essential for an efficient integration process [27, 48] and, on the other hand, likely involved in the targeting of HIV-1 integration into transcription units in human cells [49, 50]. Both of these characteristics make LEDGF/p75 a potential candidate for the observed peri-integrational deficits in mouse T-cells. Interestingly, murine and human LEDGF/p75 are highly homologous and all known functional regions are 100% conserved. Embryonal fibroblasts from Psip1/Ledgf knockout mice displayed severely reduced levels of HIV-1 vector integration . Of note, in this isogenic mouse context, a ~10-fold reduction in HIV-1 integrants coincided with a modest, ~1.5-fold increase in 2-LTR circles, consistent with the relative constellation seen for these two HIV-1 cDNA species in the cross-species comparison in the majority of our experiments. Furthermore, a critical role of mouse LEDGF/p75 in gene-specific integration was demonstrated in null cells both by mapping of residual integration sites and the observation of reduced transgene expression levels per integrant . Consequently, it could be highly informative to determine and potentially manipulate levels of endogenous LEDGF/p75 in restricted mouse T-cells.
Additional cellular proteins of interest include emerin and barrier-to-autointegration factor, which have been proposed to contribute to the appropriate nuclear localization of the viral DNA for chromatin engagement prior to integration in human cells , although this is still controversial . Of potential relevance for a scenario involving an inhibitory activity, Zhang et al. identified p21Waf1/Cip1/Sdi1 as a cellular factor that can influence the sensitivity of human hematopoietic precursors for infection by HIV-1 at a post-entry step . Specifically, expression of p21 in these human cells altered the fate of HIV-1 cDNA in the nucleus, apparently promoting the formation of episomal 2-LTR circles at the expense of integration.
Mice and rats are the prime candidates for the development of an immunocompetent, multi-transgenic small animal model. Primary target cells from both rodents share the inability to support virion entry and efficient transcription, and these limitations can be overcome by transgenic expression of the HIV-1 receptor complex [1, 2, 35] or human cyclin T1 ([6, 14], and C.G. and O.T.K., unpublished observation), respectively. Contrasting the severe early-phase restriction in mouse T-cells, rat T-cells efficiently support all steps of the HIV-1 replication cycle up to and including integration . Thus, mice appear to impose at least one additional replication barrier compared to rats which highlights the complexity of the task to develop genetically altered mice that are fully permissive for HIV-1 infection. The here identified restriction at the level of integration in mouse T-cells will facilitate the identification of critical host factors towards this goal.
Female BALB/c mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Animals were kept in the central animal facility of the University of Heidelberg.
The mouse T lymphoma cell lines S1A.TB.4.8.2 (S1A.TB) (TIB-27; BALB/c strain), TIMI.4 (TIB-37; C57BL/6 strain) and R1.1 (TIB-42; C58.J strain) were obtained from the American Type Culture Collection and cultivated in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum or horse serum (R1.1), 1% penicillin-streptomycin, and 1% L-glutamine (all from GIBCO). Human T-cell lines MT-4 and Jurkat, the adherent cell lines TZM-bl, 293T, HeLaint, and mouse fibroblast cell line NIH3T3 cells (CRL-1658; NIH/Swiss strain) were cultivated as reported [6, 7, 53–55]. Primary T-cells from Ficoll-gradient purified peripheral blood mononuclear cells from healthy human donors were activated by phytohaemagglutinin-P (1 μg/ml) and human recombinant interleukin-2 (IL-2) (20 nM, BioMol, Germany) as reported [25, 35, 53, 56]. Primary T-cells from BALB/c mice were prepared from spleens, which had been removed aseptically from sacrificed animals. Single-cell suspensions were prepared by pushing tissue pieces through a nylon mesh screen (70-μm-pore-size nylon, Becton Dickinson). The cell suspension was washed once with PBS and erythrocytes were lysed for 3 min with ACK lysis buffer, followed by another PBS wash. Single-cell splenocyte suspensions were cultivated in supplemented medium and activated with Concanavalin A (1 μg/ml) and IL-2 as reported , yielding proliferating cultures containing ~90% CD3-positive T-cells.
Cellular and viral DNAs were extracted using Qiagen DNeasy Tissue Kits®. Samples for quantitative HIV-1 integration PCR were extracted according to the method of Hirt [57, 58]. Briefly, 5 × 105 cells were pelleted and carefully resuspended in 160 μl HIRT Solution I. Next, 10 μl Proteinase K (20 mg/ml) and 200 μl HIRT Solution II were added and the cells were incubated for 30 min at 56°C. Then, 100 μl of 5 M sodium chloride were added to the reaction prior to overnight incubation at 4°C. Afterwards, chromosomal DNA was pelleted and the supernatant removed. The HIRT pellets were then extracted with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated in the presence of glycogen, and washed in 70% ethanol prior to resuspending the DNA pellet in elution buffer (Qiagen).
VSV-G pseudotyped stocks of a three-plasmid HIV-1-based GFP vector  (HIV-CMV-GFP) or of HIV-1NL4-3 E- GFP , carrying BlaM-Vpr, were generated in 293 T cells by calcium phosphate DNA precipitation in principle as described [6, 7]. Virus stocks were characterized for p24 CA concentration by antigen enzyme-linked immunosorbent assay and for infectious titer on TZM-bl cells, respectively . Virus stocks were treated with DNase (Turbo DNase, Ambion, Dresden, Germany) for 1 h at 37°C (5 IU per 10 μl of concentrated virus stock) to reduce the degree of plasmid contamination prior to challenge of target cells.
HIV-1 virion-fusion assay and gene expression analysis
Target T-cells (5 × 106) were left untreated or pretreated with efavirenz (100 μM, Bristol-Myers Squibb, Uxbridge, UK) for 1 h, or a hybridoma supernatant containing anti-VSV-G monoclonal antibody I1  for 15 min at 37°C. Subsequently, cells were infected with BlaM-Vpr-loaded VSV-G HIV-1 for 6 h, washed and stained overnight with CCF2/AM dye. Fusion was monitored with a three-laser BD FACSAria Cell Sorting System (Becton Dickinson, San Jose, CA) as reported [6, 22, 23, 25]. On day 3 p.i., the percentage of GFP-positive cells was measured in the identical cultures on a FACSCalibur using BD CellQuest Pro 4.0.2 Software (BD Pharmingen).
Quantification of total HIV-1 cDNA and 2-LTR circles
Levels of total HIV-1 cDNA and 2-LTR circles in infected cell lines were measured by quantitative PCR using the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) as reported in detail . For PCR standard curves, dilutions of pHIV-1NL4-3 and pU3U5, respectively, covering 5 logs were used, supplemented with DNA extracted from uninfected cells. Results for HIV-1 cDNA species were normalized to the amount of cellular DNA, which was quantified in a parallel amplification of the mouse GAPDH gene or human RNaseP gene (Applied Biosystems), respectively. Genomic standards were derived from dilutions of genomic DNA extracted from uninfected cells. All samples were run in duplicate. Data analysis was performed with the 7500 System Software (Applied Biosystems). Of note, for all HIV-1 cDNA analyses values obtained for parallel infections of efavirenz-treated cultures were subtracted from values of untreated cultures to account for residual proviral plasmid contamination in DNAse-treated virus inocula. For total HIV-1 cDNA analyses shown in this manuscript for VSV-G HIV-CMV-GFP (Figs. 1, 2, 3, 4, 5, 6) or VSV-G HIV-1NL4-3 GFP (Figs. 7, 8), the subtracted signals ranged between 0.07 – 0.65% or 0.3 – 4.6% of the untreated infection, respectively. However, in several experiments with other VSV-G HIV-1NL4-3 GFP stocks this background signal reached levels above 60% (despite DNAse treatment), not allowing a reliable quantification of total HIV-1 cDNA or integrants.
Quantification of integrated HIV-1 cDNA
The strategy to quantify HIV-1 integrants in the mouse genome by real-time PCR and the generation of a mouse integration standard cell line, NIH3T3int cells, was adapted from the protocol established for the rat species . Briefly, HIV-1 integrants were amplified in the first reaction by one primer annealing to the LTR (primer #1521 ), which contains a lambda-phage heel sequence at the 5'-end, and by two outward-facing primers that target the highly redundant consensus sequence within the mouse B1 gene  (primers #2194, 5'-ACAGCCAGGGCTACACAGAG-3' and #2231, 5'-CCTCCCAAGTGCTGGGATTAAAG-3'). The first-round amplicon was then amplified in a second reaction with a lambda-specific primer (primer #1522 ) and an LTR primer (primer #1523, ) and an LTR-specific probe (probe #1524, ). The second-round cycling conditions were identical to those used to determine the total amounts of HIV-1 cDNA and 2-LTR circles . For the detection of HIV-1 integrants in infected human cells, the conditions and reagents were identical to those reported . For each integration PCR, a specificity control reaction was run in parallel in which the cellular primer pair was omitted during the first round reaction and this value was subtracted from the total signal (not more than 20% of the absolute signal). For integration standard curves, dilutions of genomic DNA derived from NIH3T3int and HeLaint cells covering 3.1 logs were used (see also Fig. 4B). Criteria for standard validity included the slope, which indicates the efficiency of the PCR reaction within a defined copy range using diluted cellular DNA carrying defined amounts of integrated provirus (ideally 3.32, corresponding to a 100% efficient reaction). In addition, only integration PCR assays in which the standard reached a R2 coefficient > 0.9 were analyzed. The lowest detection standard of the PCR was 0.04 and 0.12 HIV-1 integrants per ng DNA for integrated provirus in mouse and human genomic DNA, respectively.
We thank Dr. Hans-Georg Kräusslich for support. We are grateful to Drs. Leo Lefrancois, Nathaniel Landau, and Didier Trono for the kind gift reagents. We thank Mrs. Julia Lenz, Drs. Monika Langlotz, Blanche Schwappach, and Viktor Sourjik for TBD FACSAria analyses. C.G. is recipient of a fellowship from the Peter and Traudl Engelhorn-Stiftung. This work was in part supported by the European ExCellENT-HIT consortium (LSH-CT-2006-037257) to O.T.K.
- Browning J, Horner JW, Pettoello-Mantovani M, Raker C, Yurasov S, DePinho RA, Goldstein H: Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc Natl Acad Sci USA. 1997, 94 (26): 14637-14641. 10.1073/pnas.94.26.14637.PubMed CentralView ArticlePubMedGoogle Scholar
- Sawada S, Gowrishankar K, Kitamura R, Suzuki M, Suzuki G, Tahara S, Koito A: Disturbed CD4+ T cell homeostasis and in vitro HIV-1 susceptibility in transgenic mice expressing T cell line-tropic HIV-1 receptors. J Exp Med. 1998, 187 (9): 1439-1449. 10.1084/jem.187.9.1439.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei P, Garber ME, Fang SM, Fischer WH, Jones KA: A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell. 1998, 92 (4): 451-462. 10.1016/S0092-8674(00)80939-3.View ArticlePubMedGoogle Scholar
- Bieniasz PD, Cullen BR: Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells. J Virol. 2000, 74 (21): 9868-9877. 10.1128/JVI.74.21.9868-9877.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Mariani R, Rutter G, Harris ME, Hope TJ, Krausslich HG, Landau NR: A block to human immunodeficiency virus type 1 assembly in murine cells. J Virol. 2000, 74 (8): 3859-3870. 10.1128/JVI.74.8.3859-3870.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Goffinet C, Michel N, Allespach I, Tervo HM, Hermann V, Krausslich HG, Greene WC, Keppler OT: Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression. Retrovirology. 2007, 4: 53-10.1186/1742-4690-4-53.PubMed CentralView ArticlePubMedGoogle Scholar
- Keppler OT, Yonemoto W, Welte FJ, Patton KS, Iacovides D, Atchison RE, Ngo T, Hirschberg DL, Speck RF, Goldsmith MA: Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1. J Virol. 2001, 75 (17): 8063-8073. 10.1128/JVI.75.17.8063-8073.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Landau NR, Warton M, Littman DR: The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4. Nature. 1988, 334 (6178): 159-162. 10.1038/334159a0.View ArticlePubMedGoogle Scholar
- Cohen J: Building a small-animal model for AIDS, block by block. Science. 2001, 293 (5532): 1034-1036. 10.1126/science.293.5532.1034.View ArticlePubMedGoogle Scholar
- Alonso A, Derse D, Peterlin BM: Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells. J Virol. 1992, 66 (7): 4617-4621.PubMed CentralPubMedGoogle Scholar
- Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR: Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. Embo J. 1998, 17 (23): 7056-7065. 10.1093/emboj/17.23.7056.PubMed CentralView ArticlePubMedGoogle Scholar
- Garber ME, Wei P, KewalRamani VN, Mayall TP, Herrmann CH, Rice AP, Littman DR, Jones KA: The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev. 1998, 12 (22): 3512-3527. 10.1101/gad.12.22.3512.PubMed CentralView ArticlePubMedGoogle Scholar
- Kwak YT, Ivanov D, Guo J, Nee E, Gaynor RB: Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation. J Mol Biol. 1999, 288 (1): 57-69. 10.1006/jmbi.1999.2664.View ArticlePubMedGoogle Scholar
- Sun J, Soos T, Kewalramani VN, Osiecki K, Zheng JH, Falkin L, Santambrogio L, Littman DR, Goldstein H: CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate. J Virol. 2006, 80 (4): 1850-1862. 10.1128/JVI.80.4.1850-1862.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Coskun AK, van Maanen M, Nguyen V, Sutton RE: Human chromosome 2 carries a gene required for production of infectious human immunodeficiency virus type 1. J Virol. 2006, 80 (7): 3406-3415. 10.1128/JVI.80.7.3406-3415.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Mariani R, Rasala BA, Rutter G, Wiegers K, Brandt SM, Krausslich HG, Landau NR: Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly. J Virol. 2001, 75 (7): 3141-3151. 10.1128/JVI.75.7.3141-3151.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Coskun AK, van Maanen M, Janka D, Stockton D, Stankiewicz P, Yatsenko S, Sutton RE: Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release. Virology. 2007, 362 (2): 283-293. 10.1016/j.virol.2006.12.015.PubMed CentralView ArticlePubMedGoogle Scholar
- Mariani R, Chen D, Schröfelbauer B, Navarro F, Konig R, Bollman B, Munk C, Nymark-McMahon H, Landau NR: Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell. 2003, 114 (1): 21-31. 10.1016/S0092-8674(03)00515-4.View ArticlePubMedGoogle Scholar
- Hatziioannou T, Cowan S, Bieniasz PD: Capsid-dependent and -independent postentry restriction of primate lentivirus tropism in rodent cells. J Virol. 2004, 78 (2): 1006-1011. 10.1128/JVI.78.2.1006-1011.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Baumann JG, Unutmaz D, Miller MD, Breun SK, Grill SM, Mirro J, Littman DR, Rein A, KewalRamani VN: Murine T cells potently restrict human immunodeficiency virus infection. J Virol. 2004, 78 (22): 12537-12547. 10.1128/JVI.78.22.12537-12547.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsurutani N, Yasuda J, Yamamoto N, Choi BI, Kadoki M, Iwakura Y: Nuclear Import of the Pre-integration Complex Is Blocked upon Infection by HIV-1 in Mouse Cells. J Virol. 2007, 81 (2): 677-88. 10.1128/JVI.00870-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Cavrois M, De Noronha C, Greene WC: A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol. 2002, 20 (11): 1151-1154. 10.1038/nbt745.View ArticlePubMedGoogle Scholar
- Venzke S, Michel N, Allespach I, Fackler OT, Keppler OT: Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surface of target cells and thereby enhances resistance to superinfection. J Virol. 2006, 80 (22): 11141-52. 10.1128/JVI.01556-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Boritz E, Gerlach J, Johnson JE, Rose JK: Replication-competent rhabdoviruses with human immunodeficiency virus type 1 coats and green fluorescent protein: entry by a pH-independent pathway. J Virol. 1999, 73 (8): 6937-6945.PubMed CentralPubMedGoogle Scholar
- Goffinet C, Allespach I, Keppler OT: HIV-susceptible transgenic rats allow rapid preclinical testing of antiviral compounds targeting virus entry or reverse transcription. Proc Natl Acad Sci USA. 2007, 104 (3): 1015-1020. 10.1073/pnas.0607414104.PubMed CentralView ArticlePubMedGoogle Scholar
- Jacque JM, Stevenson M: The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity. Nature. 2006, 441 (7093): 641-645. 10.1038/nature04682.View ArticlePubMedGoogle Scholar
- Llano M, Saenz DT, Meehan A, Wongthida P, Peretz M, Walker WH, Teo W, Poeschla EM: An essential role for LEDGF/p75 in HIV integration. Science. 2006, 314 (5798): 461-464. 10.1126/science.1132319.View ArticlePubMedGoogle Scholar
- Brussel A, Sonigo P: Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus. J Virol. 2003, 77 (18): 10119-10124. 10.1128/JVI.77.18.10119-10124.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J: Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 2005, 110 (1–4): 462-467. 10.1159/000084979.View ArticlePubMedGoogle Scholar
- Leavitt AD, Robles G, Alesandro N, Varmus HE: Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol. 1996, 70 (2): 721-728.PubMed CentralPubMedGoogle Scholar
- Feng Y, Broder CC, Kennedy PE, Berger EA: HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996, 272 (5263): 872-877. 10.1126/science.272.5263.872.View ArticlePubMedGoogle Scholar
- Sheehy AM, Gaddis NC, Malim MH: The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med. 2003, 9 (11): 1404-1407. 10.1038/nm945.View ArticlePubMedGoogle Scholar
- Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J: The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature. 2004, 427 (6977): 848-853. 10.1038/nature02343.View ArticlePubMedGoogle Scholar
- Hatziioannou T, Princiotta M, Piatak M, Yuan F, Zhang F, Lifson JD, Bieniasz PD: Generation of simian-tropic HIV-1 by restriction factor evasion. Science. 2006, 314 (5796): 95-10.1126/science.1130994.View ArticlePubMedGoogle Scholar
- Keppler OT, Welte FJ, Ngo TA, Chin PS, Patton KS, Tsou CL, Abbey NW, Sharkey ME, Grant RM, You Y, Scarborough JD, Ellmeier W, Littman DR, Stevenson M, Charo IF, Herndier BG, Speck RF, Goldsmith MA: Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1. J Exp Med. 2002, 195: 719-736. 10.1084/jem.20011549.PubMed CentralView ArticlePubMedGoogle Scholar
- Butler SL, Johnson EP, Bushman FD: Human immunodeficiency virus cDNA metabolism: notable stability of two-long terminal repeat circles. J Virol. 2002, 76 (8): 3739-3747. 10.1128/JVI.76.8.3739-3747.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Li L, Olvera JM, Yoder KE, Mitchell RS, Butler SL, Lieber M, Martin SL, Bushman FD: Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. Embo J. 2001, 20 (12): 3272-3281. 10.1093/emboj/20.12.3272.PubMed CentralView ArticlePubMedGoogle Scholar
- Pierson TC, Kieffer TL, Ruff CT, Buck C, Gange SJ, Siliciano RF: Intrinsic stability of episomal circles formed during human immunodeficiency virus type 1 replication. J Virol. 2002, 76 (8): 4138-4144. 10.1128/JVI.76.8.4138-4144.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Sharkey ME, Teo I, Greenough T, Sharova N, Luzuriaga K, Sullivan JL, Bucy RP, Kostrikis LG, Haase A, Veryard C, Davaro RE, Cheeseman SH, Daly JS, Bova C, Ellison RT, Mady B, Lai KK, Moyle G, Nelson M, Gazzard B, Shaunak S, Stevenson M: Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nat Med. 2000, 6: 76-81. 10.1038/71569.View ArticlePubMedGoogle Scholar
- Jurka J, Kohany O, Pavlicek A, Kapitonov VV, Jurka MV: Clustering, duplication and chromosomal distribution of mouse SINE retrotransposons. Cytogenet Genome Res. 2005, 110 (1–4): 117-123. 10.1159/000084943.View ArticlePubMedGoogle Scholar
- Schröder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F: HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002, 110 (4): 521-529. 10.1016/S0092-8674(02)00864-4.View ArticlePubMedGoogle Scholar
- Shun MC, Raghavendra NK, Vandegraaff N, Daigle JE, Hughes S, Kellam P, Cherepanov P, Engelman A: LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 2007, 21 (14): 1767-1778. 10.1101/gad.1565107.PubMed CentralView ArticlePubMedGoogle Scholar
- Cherepanov P, Maertens G, Proost P, Devreese B, Van Beeumen J, Engelborghs Y, De Clercq E, Debyser Z: HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J Biol Chem. 2003, 278 (1): 372-381. 10.1074/jbc.M209278200.View ArticlePubMedGoogle Scholar
- Maertens G, Cherepanov P, Pluymers W, Busschots K, De Clercq E, Debyser Z, Engelborghs Y: LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J Biol Chem. 2003, 278 (35): 33528-33539. 10.1074/jbc.M303594200.View ArticlePubMedGoogle Scholar
- Van Maele B, De Rijck J, De Clercq E, Debyser Z: Impact of the central polypurine tract on the kinetics of human immunodeficiency virus type 1 vector transduction. J Virol. 2003, 77 (8): 4685-4694. 10.1128/JVI.77.8.4685-4694.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Busschots K, Vercammen J, Emiliani S, Benarous R, Engelborghs Y, Christ F, Debyser Z: The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding. J Biol Chem. 2005, 280 (18): 17841-17847. 10.1074/jbc.M411681200.View ArticlePubMedGoogle Scholar
- Emiliani S, Mousnier A, Busschots K, Maroun M, Van Maele B, Tempe D, Vandekerckhove L, Moisant F, Ben-Slama L, Witvrouw M, Christ F, Rain JC, Dargemont C, Debyser Z, Benarous R: Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication. J Biol Chem. 2005, 280 (27): 25517-25523. 10.1074/jbc.M501378200.View ArticlePubMedGoogle Scholar
- Vandekerckhove L, Christ F, Van Maele B, De Rijck J, Gijsbers R, Haute Van den C, Witvrouw M, Debyser Z: Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus. J Virol. 2006, 80 (4): 1886-1896. 10.1128/JVI.80.4.1886-1896.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Ciuffi A, Llano M, Poeschla E, Hoffmann C, Leipzig J, Shinn P, Ecker JR, Bushman F: A role for LEDGF/p75 in targeting HIV DNA integration. Nat Med. 2005, 11 (12): 1287-1289. 10.1038/nm1329.View ArticlePubMedGoogle Scholar
- Poeschla EM: Integrase, LEDGF/p75 and HIV replication. Cell Mol Life Sci. 2008Google Scholar
- Shun MC, Daigle JE, Vandegraaff N, Engelman A: Wild-type levels of human immunodeficiency virus type 1 infectivity in the absence of cellular emerin protein. J Virol. 2007, 81 (1): 166-172. 10.1128/JVI.01953-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang J, Scadden DT, Crumpacker CS: Primitive hematopoietic cells resist HIV-1 infection via p21. J Clin Invest. 2007, 117 (2): 473-481. 10.1172/JCI28971.PubMed CentralView ArticlePubMedGoogle Scholar
- Keppler OT, Allespach I, Schüller L, Fenard D, Greene WC, Fackler OT: Rodent cells support key functions of the human immunodeficiency virus type 1 pathogenicity factor Nef. J Virol. 2005, 79 (3): 1655-1665. 10.1128/JVI.79.3.1655-1665.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y: Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature. 1981, 294 (5843): 770-771. 10.1038/294770a0.View ArticlePubMedGoogle Scholar
- Pauwels R, De Clercq E, Desmyter J, Balzarini J, Goubau P, Herdewijn P, Vanderhaeghe H, Vandeputte M: Sensitive and rapid assay on MT-4 cells for detection of antiviral compounds against the AIDS virus. J Virol Methods. 1987, 16 (3): 171-185. 10.1016/0166-0934(87)90002-4.View ArticlePubMedGoogle Scholar
- Goffinet C, Keppler OT: Efficient nonviral gene delivery into primary lymphocytes from rats and mice. Faseb J. 2006, 20 (3): 500-502.PubMedGoogle Scholar
- Vandegraaff N, Kumar R, Burrell CJ, Li P: Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA. J Virol. 2001, 75 (22): 11253-11260. 10.1128/JVI.75.22.11253-11260.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Hirt B: Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967, 26 (2): 365-369. 10.1016/0022-2836(67)90307-5.View ArticlePubMedGoogle Scholar
- Zufferey R, Donello JE, Trono D, Hope TJ: Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 1999, 73 (4): 2886-2892.PubMed CentralPubMedGoogle Scholar
- He J, Chen Y, Farzan M, Choe H, Ohagen A, Gartner S, Busciglio J, Yang X, Hofmann W, Newman W, Mackay CR, Sodroski J, Gabuzda D: CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature. 1997, 385 (6617): 645-649. 10.1038/385645a0.View ArticlePubMedGoogle Scholar
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