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T-cell acute lymphoblastic leukemia is a high-risk type of blood-cell cancer. We analyzed the possibility of developing virotherapy for T-cell acute lymphoblastic leukemia. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. We constructed a minimized derivative of HIV-1, a complex lentivirus encoding multiple accessory functions that are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. This mini-HIV virus has five deletions (vif, vpR, vpU, nef, and U3) and replicated in the SupT1 cell line, but did not replicate in normal peripheral blood mononuclear cells. The stripped down mini-HIV variant was also able to efficiently remove leukemic cells from a mixed culture with untransformed control cells. In contrast to wild-type HIV-1, we did not observe bystander killing in mixed culture experiments with the mini-HIV variant. Furthermore, viral escape was not detected in long-term cultures. The mini-HIV variant that uses CD4 and CXCR4 for cell entry could potentially be used against CXCR4-expressing malignancies such as T-lymphoblastic leukemia/lymphoma, natural killer leukemia, and some myeloid leukemias.

Supplementary Figure 1 from Construction of a Minimal HIV-1 Variant that Selectively Replicates in Leukemic Derived T-Cell Lines: Towards a New Virotherapy Approach

One of the main challenges in early clinical research with respiratory syncytial virus (RSV) live-attenuated vaccines (LAVs) is to assess immunogenicity in healthy adults. Healthy adults will have preexisting levels of serum neutralizing antibodies that could prematurely neutralize the LAV and underestimate the potential effect of the vaccine on the immune system. Data on prevalence and distribution of virus neutralizing titers (VNTs) in healthy adults is limited and there is no absolute threshold for protection against RSV-infection that can serve as an eligibility criterion in early phase trials. We assessed the RSV-specific serum VNT in healthy adults outside the Dutch RSV-Season in two clinical studies performed in 2017 (exploratory study, n = 100) and 2018 (first-in-human LAV-study, n = 190) using the same neutralizing assay. Our findings show that the prevalence and distribution of serum VNT was overall consistent in the two clinical studies. Log2 VNTs were normally distributed, distributions of VNTs were similar and there was no statistical difference in mean log2 VNT for both studies (p = .3). Serum VNTs were comparable during the 6 months of screening in the FIH LAV-study. Our findings will help to determine a cutoff serum VNT to be used as an eligibility criterion in future early phase clinical trials.

Background: VIS410, a broadly neutralizing monoclonal antibody that binds the hemagglutinin stem of influenza A viruses, was safe and efficacious in a human H1N1 virus challenge study. This study evaluated the safety and tolerability of VIS410 in non-hospitalized adult patients with uncomplicated influenza A. Methods: Patients 18 to 65 years of age with symptom onset within 72 h were randomized 1:1:1 to receive a single intravenous infusion of VIS410 4000 mg, 2000 mg, or placebo. Neuraminidase inhibitor therapy was prohibited. Treatment-emergent adverse events (TEAEs) were evaluated up to 100 days post-infusion. Influenza symptoms were assessed daily for 10 days using the FLU-PRO tool. Nasopharyngeal virus shedding was assessed by quantitative reverse-transcription PCR (qRT-PCR) and viral culture through Day 7. Findings: Of the 150 patients randomized, 148 received study drug, and 138 were confirmed influenza A positive. Median age was 42 years; median time from symptom onset to treatment was 42 h; 93% had influenza A subtype H3N2. Safety: TEAEs, most commonly diarrhea of mild severity, were dose-related, occurring in 55%, 35%, and 24% of the 4000 mg, 2000 mg, and placebo patients, respectively. Two serious adverse events occurred, both in placebo patients. Symptom analyses: Baseline FLU-PRO symptom scores were balanced among groups. Mean scores were lower by Days 3 and 4 in the pooled VIS410 treatment group versus placebo (p

Background The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. Methods Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. Findings Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR + DC-stimulation reversed latency more frequently. Such a “one-two punch” strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. Interpretation This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we demonstrate that TCR-stimulated effector T cells still frequently harbor latent HIV-1. Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with dendritic cells (DCs) triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR+DC-stimulation reversed latency more frequently. Such a “one-two punch” strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates.

HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.

Avian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.

Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon.

Treatment of an HIV infected individual with antiretroviral drugs is a successful way to suppress the plasma viral RNA load below the limit of detection (50 copies HIV RNA/ml plasma). This can provide lifelong protection against virus-induced pathogenesis in drug-adherent patients. Unfortunately, even after many years of continuous treatment, the virus persists and the plasma viral load will rebound rapidly when therapy is interrupted. The reason for this rapid rebound is the presence of a long-lived reservoir of latent HIV-1 proviruses that can be reactivated in resting memory T cells. Attempts to eliminate these proviruses have thus far not been successful and this long-lived latent reservoir is therefore considered a major obstacle toward a cure for HIV-1. A detailed understanding of the molecular mechanisms causing HIV latency and knowledge on the establishment of this reservoir may give us clues for future strategies aiming at the eradication of this reservoir.

In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected. (C) 2010 Elsevier B.V. All rights reserved

Background: Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro. Methods: Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir. Results: Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells’ ability to induce viral gene expression. Conclusions: In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies. Conclusions: Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation.

The latest World Health Organization (WHO) figures (June 17, 2009) show that 88 countries have officially reported 39,620 cases of influenza A (H1N1) infection, including 167 deaths.1 The rapid worldwide spread and the apparent lack of preexisting immunity in humans qualify this as a pandemic virus. The identification of this novel virus in the beginning of April 2009 and subsequent international screening programs have demonstrated that the virus is able to spread from human to human. These data are sufficient to state that we are currently experiencing an influenza pandemic. WHO raised the pandemic influenza alert to phase 6 on June 10, 2009.

Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5′ end of the viral RNA (Fig. 1 Fig. 1. Template RNA structure of the HIV-1 R region and the position of reverse transcription pause sites. The HIV-1 R region (nucleotides +1/97) encodes two stable RNA structures, the TAR and polyA hairpins [5]. The latter hairpin contains the AAUAAA hexamer motif (marked by a box) that is involved in polyadenylation. The lower panel shows the predicted structures of the wild-type and two mutant forms of the polyA hairpin that were used in this study. Nucleotide substitutions are boxed, deletions are indicated by ▴. The thermodynamic stability (free energy or ΔG, in kcal/mol) was calculated according to the Zucker algorithm [71]. The TAR hairpin has a ΔG of −24.8 kcal/mol. Minus-strand DNA synthesis on these templates was initiated by a DNA primer annealed to the downstream PBS. The position of reverse transcription pause sites observed in this study are summarized. All numbers refer to nucleotide positions on the wild-type HIV-1 transcript. Filled arrows represent stops observed on the wild-type template, and open arrows mark the pause sites that are specific for the structured A-mutant template. The sizes of the arrows correspond to the relative frequency of pausing. Little pausing was observed on the B-mutant template with the destabilized polyA hairpin. Figure options Download full-size image Download high-quality image (242 K) Download as PowerPoint slide ). These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg2+ concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription.

Laboratory-confirmed cases of subclinical infection with avian influenza A(H5N1) virus in humans are rare, and the true number of these cases is unknown. We describe the identification of a laboratory-confirmed subclinical case in a woman during an influenza A(H5N1) contact investigation in northern Vietnam.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus., IMPORTANCE Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the RNAi pathway is necessary. Apart from the intended therapeutic effect, this treatment can induce negative effects on cell proliferation via off-target effects. A careful evaluation of the transduced cells is required to develop a safe gene therapy approach. Stably transduced cells are usually selected by expression of the enhanced green fluorescent protein (GFP) marker. In this study we show that the mixed transduction culture, containing both transduced GFP(+) and untransduced GFP(-) cells, can simply be passaged to score the GFP(+)/GFP(-) ratio by longitudinal flow cytometric analysis as a measure of the negative impact of the RNAi treatment on the cellular proliferation rate. We show that this assay is sensitive, easy to use and internally controlled for assessing subtle effects on cell proliferation of lentiviral transduction and transgene expression.

The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form.

This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of human immunodeficiency virus type 1 (HIV-1)-T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE) coupled to matrix assisted laser-desorption time of flight peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MS(E), a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1

The current HIV-1 pandemic is caused by at least nine subtypes (termed A through K) and an increasing number of recombinant forms. There has been a large skew in research focus on subtype B, which is the most prevalent subtype in the Western world. We study the transcriptional promoter in the long terminal repeat (LTR). Each subtype has a specific LTR promoter configuration and even minor changes in the transcription factor-binding sites (TFBS) or their rearrangement can have a significant impact on cell tropism and pathogenicity. For the HIV-1 subtypes, the LTR promoter variation could contribute to differences in proviral latency. Such differences in latency properties may have an impact on the establishment of viral reservoirs and the inability to clear the virus by therapeutic intervention. Latency remains a formidable barrier towards virus eradication as therapeutic attempts to purge these reservoirs have been unsuccessful. The pool of latent proviruses is established early during infection and forms a steady source of proviral DNA that can last a lifetime in infected individuals. We have demonstrated previously that there are differences in proviral latency properties between the subtypes, which are determined in part by the LTR promoter. Most interesting was the observation that subtype AE combined higher basal transcription (-Tat) with reduced proviral latency. The naturally occurring replacement of an NFkB for GABP site was partially responsible for this AE-specific latency phenotype. Another transcription factor that may contribute to the increased basal transcription of subtype AE is AP-1, which is present in subtype AE but absent in subtype B. We generated luciferase constructs with and without this AP-1 site in both subtype LTR backbones to determine the influence of AP-1. The luciferase constructs contain the core promoter region with all major TFBS (e.g. NFkB and Sp1 sites). Results obtained with these reporter constructs will be presented.

Background: HIV-1 transcription initiation depends on cellular transcription factors that bind to promoter sequences in the Long Terminal Repeat (LTR). Each HIV-1 subtype has a specific LTR promoter configuration and even minor sequence changes in the transcription factor binding sites (TFBS) or their arrangement can impact transcriptional activity. Most latency studies have focused on HIV-1 subtype B strains, and the degree to which LTR promoter variation contributes to differences in proviral latency is therefore largely unknown. Latency differences may influence establishment and size of viral reservoirs as well as the possibility to clear the virus by therapeutic intervention. Results: We investigated the proviral transcriptional latency properties of different HIV-1 subtypes as their LTRs have unique assemblies of transcription factor binding sites. We constructed recombinant viral genomes with the subtype-specific promoters inserted in the common backbone of the subtype B LAI isolate. The recombinant viruses are isogenic, except for the core promoter region that encodes all major TFBS, including NFB and Sp1 sites. We developed and optimized an assay to investigate HIV-1 proviral latency in T cell lines. Our data show that the majority of HIV-1 infected T cells only start viral gene expression after TNFa activation. Conclusions: There were no gross differences among the subtypes, both in the initial latency level and the activation response, except for subtype AE that combines an increased level of basal transcription with a reduced TNFa response. This subtype AE property is related to the presence of a GABP instead of NFB binding site in the LTR. Background Combined antiretroviral therapy (cART) is able to suppress the HIV-1 plasma RNA load in patients to undetectable levels. Unfortunately, the treatment does not lead to a complete eradication of the virus from the infected individual. Even after many years of successful cART, the virus rebounds from latently integrated proviral DNA reservoirs and re-establishes systemic infection upon interruption of therapy [1-4]. HIV-1 proviral latency may be an effective means to evade the immune system, since the infected cell will go unnoticed by the immune system as long as viral antigens are not expressed and presented. The pool of latent proviruses is established early during infection and forms a steady source of proviral DNA that can last a lifetime for infected individuals [5-7]. The majority of the

Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication-competent virus has been minimized. Nevertheless, there may be certain applications for replication-competent HIV-based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV-1 variant as a safe live-attenuated virus vaccine, the construction of mini-HIV variants as cancer-selective viruses for virotherapy against leukemia, and the use of a conditionally live anti-HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication-competent HIV-based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models.

We identified an HIV-1 variant that belongs to the M group, with limited similarity of short genetic regions (100-200 nt) to subtype K, but the remainder of the genome is unrelated to any established HIV-1 subtype. The isolate was obtained from an HIV-1-positive male, living in the Netherlands, who encountered the virus before 1989, most probably via heterosexual contact in Africa. We describe the full-length genome sequence of four biological clones that were obtained from two samples collected 5 years apart. At both time points all open reading frames were intact. Within the 5-year interval, the person received antiretroviral therapy with zalcitabine and zidovudine for almost 4 years. Evolution of drug-resistant variants is likely given the increase in viral RNA load to +/-10,000 copies/ml during the last year of treatment. Surprisingly, the only regular RT mutation acquired during this period was K70R, which suggests that the genetic background of this variant is perhaps not suitable for the generation of the standard 41L, 67N, and 215Y/F mutations that typically arise during prolonged, nonsuccessful, zidovudine treatment. Awaiting the discovery of at least two additional, epidemiologically unrelated patients with a phylogenetically related HIV-1 variant, we can designate this variant a new HIV-1 subtype, or a distinct branch of subtype K.

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3) and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.

Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-κB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.

T-cell acute lymphoblastic leukemia is a high-risk type of blood-cell cancer. We analyzed the possibility of developing virotherapy for T-cell acute lymphoblastic leukemia. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. We constructed a minimized derivative of HIV-1, a complex lentivirus encoding multiple accessory functions that are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. This mini-HIV virus has five deletions (vif, vpR, vpU, nef, and U3) and replicated in the SupT1 cell line, but did not replicate in normal peripheral blood mononuclear cells. The stripped down mini-HIV variant was also able to efficiently remove leukemic cells from a mixed culture with untransformed control cells. In contrast to wild-type HIV-1, we did not observe bystander killing in mixed culture experiments with the mini-HIV variant. Furthermore, viral escape was not detected in long-term cultures. The mini-HIV variant that uses CD4 and CXCR4 for cell entry could potentially be used against CXCR4-expressing malignancies such as T-lymphoblastic leukemia/lymphoma, natural killer leukemia, and some myeloid leukemias.

The human immunodeficiency virus type 1 (HIV-1) transcriptional promoter contains a single polymorphism in the TATA box. Most subtypes contain the sequence TATAAGC, but subtype E and some recombinant AG strains have the sequence TA A AAGC. Based on mutagenesis studies of cellular RNA polymerase II (pol II) promoters, it has been proposed that the subtype E TATA box is nonfunctional due to the T-to-A substitution at the critical position 3. By means of transcription and virus replication assays, we demonstrate that the true TATA box motif within the viral long terminal repeat (LTR) promoter starts two nucleotides further upstream. Because of this realignment, subtype E has the sequence CA TA A AA and all other subtypes have the sequence CA TATAA. The polymorphism therefore has shifted from position 3 to position 5 and is no longer incompatible with efficient transcription according to rules determined for cellular pol II promoters. In addition, through sensitive competition experiments, we demonstrate that the CATA box of subtypes B and E can be improved for replication by the mutations 1T and 5T, respectively. The fact that the fitness of both subtype LTRs can be increased by specific point mutations in the CATA box suggests that the transcriptional promoter of HIV-1 is fine-tuned towards a suboptimal level of replication. However, this replication rate may be optimal in the in vivo context of an infected individual.

The long terminal repeat (LTR) transcriptional promoters of different human immunodeficiency virus (HIV) type 1 subtypes were inserted into the LAI molecular clone of subtype B. The viral genotypes represent seven subtypes (A, B, C, D, E, F, and G) and one circulating recombinant form (AG). We performed replication studies with this isogenic set of viruses across six cellular environments. This approach revealed strong cellular environment effects, but the method was not sensitive enough to detect small differences in the replication rate between the subtypes. By conducting pairwise competition experiments between the virus variants in six cellular environments, we could demonstrate significant differences in the replication rates of the subtypes and that LTR-determined viral fitness depends both on the host cell type and the activation state of the cell. In addition, we determined the degree of conservation of the transcription factor-binding sites (TFBS) in the different-subtype LTRs by analyzing sequences from the HIV sequence database. The sequence analyses revealed subtype-specific conservation of certain TFBS. The results indicate that one should consider the possibility of subtype-specific viral replication rates in vivo, which are strongly influenced by the host environment. We argue that the multidimensional host environment may have shaped the genetic structures of the subtype LTRs.

The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

The human immunodeficiency virus type 1 (HIV-1) is notorious for its ability to evolve drug-resistance in patients treated with potent antivirals. Resistance to inhibitors of the viral reverse transcriptase (RT) enzyme is frequently mediated by a single amino acid substitution within RT. Resistance against the nucleoside analogue AZT is remarkable in that multiple amino acid changes accumulate over time to yield virus variants with high-level drug resistance. We now report that in addition to drug-resistance properties, the relative replication capacity of the virus variants affects the evolution of AZT resistance. Some of the typical AZT-resistance mutations have a negative impact on virus replication, and the 41-70 double mutant was found to represent a particularly poor virus. Furthermore, introduction of additional AZT-resistance mutations (41-70-215) leads to nearly complete restoration of virus replication. These results may explain the absence of the 41-70 double mutant in clinical samples and indicate that the evolution of AZT resistance is also influenced by virus replication parameters. Prolonged passage of the replication-impaired 41-70 virus in the absence of AZT yielded several fast-replicating variants. These revertants have compensatory changes in the RT polymerase, some of which have been observed previously in AZT-treated patients. Because we could select for these changes without drug pressure, these changes are likely to improve the RT enzyme function and the HIV-1 replication capacity.

The current human immunodeficiency virus type 1 (HIV-1) shows an increasing number of distinct viral subtypes, as well as viruses that are recombinants of at least two subtypes. Although no biological differences have been described so far for viruses that belong to different subtypes, there is considerable sequence variation between the different HIV-1 subtypes. The HIV-1 long terminal repeat (LTR) encodes the transcriptional promoter, and the LTR of subtypes A through G was cloned and analyzed to test if there are subtype-specific differences in gene expression. Sequence analysis demonstrated a unique LTR enhancer-promoter configuration for each subtype. Transcription assays with luciferase reporter constructs showed that all subtype LTRs are functional promoters with a low basal transcriptional activity and a high activity in the presence of the viral Tat transcriptional activator protein. All subtype LTRs responded equally well to the Tat trans activator protein of subtype B. This result suggests that there are no major differences in the mechanism of Tat-mediated trans activation among the subtypes. Nevertheless, subtype-specific differences in the activity of the basal LTR promoter were measured in different cell types. Furthermore, we measured a differential response to tumor necrosis factor alpha treatment, and the induction level correlated with the number of NF-κB sites in the respective LTRs, which varies from one (subtype E) to three (subtype C). In general, subtype E was found to encode the most potent LTR, and we therefore inserted the core promoter elements of subtype E in the infectious molecular clone of the LAI isolate (subtype B). This recombinant LAI-E virus exhibited a profound replication advantage compared with the original LAI virus in the SupT1 T-cell line, indicating that subtle differences in LTR promoter activity can have a significant impact on viral replication kinetics. These results suggest that there may be considerable biological differences among the HIV-1 subtypes.

The mechanism of reverse transcription was analyzed in vitro with RNA templates and the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). In particular, we analyzed the mechanism of actinomycin D (ActD) mediated inhibition of the strand transfer step, in which the newly synthesized cDNA, termed the (-) strand strong stop or (-)ssDNA, is transferred from the donor RNA onto the acceptor RNA. This strand transfer reaction is a rather inefficient process in vitro. We found that this is in part due to the presence of an excess donor RNA, and highly efficient strand transfer was achieved by reducing the amount of donor RNA. We suggest that annealing of the (-)ssDNA to the excess donor RNA is preferred over productive binding to the acceptor RNA because of a higher basepair complementarity. ActD remains a potent inhibitor of strand transfer in this optimized assay system. We measured no effect of ActD on the elongation of reverse transcription or the RNase H action of the RT enzyme. Instead, we provide evidence that ActD acts through direct interaction with the (-)ssDNA, thereby blocking the basepairing capacity of this molecule. The possible use of single-stranded DNA binding molecules as antiretroviral agents is discussed.

HIV-1 latency remains a formidable barrier towards virus eradication as therapeutic attempts to purge these reservoirs are so far unsuccessful. The pool of transcriptionally silent proviruses is established early in infection and persists for a lifetime, even when viral loads are suppressed below detection levels using anti-retroviral therapy. Upon therapy interruption the reservoir can re-establish systemic infection. Different cellular reservoirs that harbor latent provirus have been described. In this study we demonstrate that HIV-1 can also establish a silent integration in actively proliferating primary T lymphocytes. Co-culturing of these proliferating T lymphocytes with dendritic cells (DCs) activated the provirus from latency. Activation did not involve DC-mediated C-type lectin DC-SIGN signaling or TCR-stimulation but was mediated by DC-secreted component(s) and cell-cell interaction between DC and T lymphocyte that could be inhibited by blocking ICAM-1 dependent adhesion. These results imply that circulating DCs could purge HIV-1 from latency and re-initiate virus replication. Moreover, our data show that viral latency can be established early after infection and supports the idea that actively proliferating T lymphocytes with an effector phenotype contribute to the latent viral reservoir. Unraveling this physiologically relevant purging mechanism could provide useful information for the development of new therapeutic strategies that aim at the eradication of HIV-1 reservoirs., Author Summary Combination therapy can suppress the viral load in HIV-1 infected individuals to undetectable levels, but does not lead to complete virus eradication. Even after many years of successful therapy the virus is still present in long-lived cells as a latently integrated provirus. HIV-1 can re-establish systemic infection from this reservoir when therapy stops. Purging attempts in patients have been unsuccessful and HIV-1 latency remains a formidable barrier to virus eradication. Different cellular reservoirs that harbor latent HIV-1 proviruses have been described to consist mainly of resting memory T lymphocytes. Yet how this reservoir in memory T lymphocytes is established is still unclear as infection of these cells is very inefficient. In this paper we demonstrate that HIV-1 can establish a latent provirus in activated effector T lymphocytes. We observed that for every virus producing cell there is at least one other cell harboring a latent provirus, illustrating that latent infections occur frequently. Proliferating T lymphocytes are generally short-lived and their contribution to the total cellular reservoir thus seems limited. However, these activated T lymphocytes can revert into resting memory T lymphocytes and become part of the long-lived viral reservoir.

RNAi is a sequence-specific gene silencing mechanism induced by double-stranded RNA and a promising tool for the development of a durable gene therapy against HIV-1. Such RNAi attack usually focuses on targets within the HIV-1 RNA genome. Despite potent inhibition, we previously observed the rapid selection of RNAi-resistant HIV-1 variants with a mutated target sequence. A number of strategies were tested to prevent viral escape. Similar to the success of combination therapy with multiple anti-retroviral drugs, one can target HIV-1 with multiple short hairpin RNAs (shRNAs). Alternatively, one can design second-generation shRNAs to target favorite RNAi-resistance mutations in order to block specific viral escape routes. One could also combine RNAi with regular antiviral drug regimens. Another strategy involves the use of RNAi against cellular proteins that are involved in HIV-1 replication. The idea is that viral escape is more difficult in this setting. Many cellular co-factors have been described in literature, e.g. based on large RNAi-screens in cell lines. We have shown that long-term inhibition of HIV-1 replication is possible with RNAi against cellular co-factors. To identify relevant co-factors that are not essential for cellular function, primary cells are to be preferred over T cell lines. We propose that RNAi against both viral and cellular targets could be used as a new therapeutic approach in the combat against HIV-1 infection.