A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors.

The Tat function of the human immunodeficiency virus (HIV) represents an important target for the development of new anti-HIV drugs. A rapid, sensitive and simple bioassay was developed for the detection of HIV transactivation inhibitors. A reporter plasmid based on the expression of the green fluorescent protein (GFP) under control of the HIV-1 long terminal repeat (LTR) was constructed. This reporter gene can be quantified by simply measuring the fluorescence irradiated by GFP-producing cells, without the need of extraction procedures or enzymatic assays. Cells, stably expressing HIV-1 Tat protein, were transfected with this plasmid and the inhibitory effect of anti-Tat drugs was assessed by measuring the inhibition of fluorescence. Using this assay system the anti-transactivation activity of several known compounds was confirmed. This is the first HIV transactivation assay using GFP reporter gene in microtiter plates. The assay can be used for the detection and quantification of HIV transactivation, and for the high throughput evaluation of anti-transactivation drugs in different cellular backgrounds.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

A macrophage fusion assay for rapid screening of cloned HIV-1 Env using dual recombinant vaccinia viruses expressing distinct RNA polymerases.

HIV-1 cell tropism is determined initially at the level of fusion mediated by the viral envelope glycoprotein (Env). Cell-cell fusion assays are employed widely to study Env-mediated fusion, and generally require transfection of target cells with a reporter plasmid that is activated upon fusion with Env-expressing effector cells. Macrophages are an important target for HIV-1, but fusion studies using primary macrophages are limited by their resistance to transfection. An assay described previously used recombinant vaccinia virus to express T7 polymerase in macrophages, and effector cells transfected with a T7-driven reporter plasmid and infected with recombinant vaccinia virus expressing Env. However, this requires a recombinant vaccinia virus for each Env. We developed a method to study fusion using primary macrophages and HIV-1 env plasmid clones under control of the T7 promoter. Macrophages were infected with a recombinant vaccinia virus expressing the SP6 RNA polymerase. Effector 293T cells were infected with a recombinant vaccinia virus expressing T7 polymerase, and co-transfected with T7-driven env plasmids and an SP6-driven reporter gene plasmid. Cell-cell fusion mediated by T7-driven Env results in SP6-driven reporter gene transactivation. This approach is suitable for rapid analysis of multiple primary isolate, chimeric, or mutant env genes cloned into plasmid vectors.     

Half-genome human immunodeficiency virus type 1 constructs for rapid production of reporter viruses

The use of reporter constructs of HIV-1 molecular clones has been useful in studying the effects of infection on individual cells. A strategy of producing viral half-genome constructs containing reporter genes that can be used to produce recombinant complete infectious virions is described. This approach allows rapid generation of reporter and non-reporter virions from the same genetic construct, obviating the need to produce a new reporter genetic construct for each viral change to be studied. We provide an example of the utility of this system for studying the effects of Nef on infected cells.     

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.     

A novel small reporter gene and HIV-1 fitness assay

Most currently available HIV-1 reporter gene constructs are large and disrupt the nef reading frame. This report describes a novel reporter gene based on the small murine heat stable antigen (HSA) protein, which is expressed on the surface of infected cells. This HSA reporter can be inserted in the vpr reading frame, leaving nef intact. Nine amino acids from the extracellular domain of HSA are replaced with an influenza hemagglutinin (HA) antibody epitope (HSA-HA). Like the parental reporter protein, this novel reporter is expressed on the surface of infected cells. Antibodies for HSA and HA specifically detect reporter viruses with each construct, indicating disruption of the original HSA antibody epitope. Finally, a strategy is developed to detect each reporter virus by real-time PCR quantitation. The growth of viruses tagged with each reporter allows precise assessment of the relative growth of viruses differing in mutations of interest. Moreover, the availability of these reporters in either of two half-genome plasmids allows convenient production of reporter and non-reporter HIV-1 by co-transfection of appropriately paired plasmids. These paired reporter viruses offer a potentially useful standardized method for measurement of HIV-1 fitness in competition assays.     

Flow cytometric detection of viruses

Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. The highest green fluorescence intensities were obtained using SYBR Green I. DNA viruses with genome sizes between 48.5 and 300 kb could easily be detected. The fluorescence signals of the small genome-sized RNA viruses (7.4-14.5 kb) were found at the limit of detection. No significant linear relationship could be found between genome size and the green fluorescence intensity of the SYBR Green I stained virus preparations. To our knowledge, this is the first report of detecting and discriminating between a wide range of different viruses directly using flow cytometry. This rapid and precise assay represents a new and promising tool in the field of virology.     

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.     

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.     

Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs

Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.     

High throughput functional analysis of HIV-1 env genes without cloning

Functional human immunodeficiency virus type 1 (HIV-1) env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3 Delta env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple env genes from HIV-1 infected individuals.     

An anti-HIV-1 gp120 antibody expressed as an endocytotic transmembrane protein mediates internalization of HIV-1

In this study, we used HIV-1 as a model to demonstrate a novel approach for receptor-independent cell entry of virus. The heavy chain of an anti-HIV-1 gp120 antibody was engineered with endocytotic and transmembrane motifs from either the cation-independent mannose 6-phosphate receptor or the low-density lipoprotein receptor. Flow cytometry and immunofluorescence studies showed that the chimeric antibodies were expressed on the cell surface and can undergo rapid internalization. Furthermore, one of the chimeric antibodies was able to bind and internalize HIV-1. Using a luciferase reporter HIV-1, we further showed that internalized viruses could undergo replication. Therefore, we have demonstrated a proof-of-principle of a novel method that can be used to internalize virus into cells, without prior knowledge of the cellular receptor for the virus. We propose that this approach would be particularly useful for studying viruses whose cellular receptor(s) is not known.     

ASPCR检测微量HIV-1 K103N耐药突变方法的建立

目的 建立检测微量HIV-1 K103N耐药突变的等位基因特异扩增实时定量PCR(allele-specific real-time PCR,ASPCR)方法,用于微量K103N耐药突变的检测和分析.方法 首先建立检测K103N耐药突变的ASPCR方法,然后对方法进行验证,最后采用ASPCR方法检测对照样本.构建含K103N突变的质粒作为标准品,然后设计特异性引物用以区分野生型和突变型模板,采用SYBR green法进行实时定量PCR,并绘制标准曲线.分别采用特异性和非特异性引物扩增模板,根据二者Ct(cycle threshold)值结合相应的标准曲线判断是否存在K103N突变及突变比例.对ASPCR检测K103N突变位点的特异性、敏感性、准确性、重复性等进行验证.结果 特异性和非特异性引物扩增等量野生型模板的Ct值之差(△Ct)可达13.56;当突变比例小于0.1%时仍具有良好的准确性;批内变异系数小于0.7,批间变异系数小于1.6;检测灵敏度可达0.01%.检测阴性对照样本的△Ct显著高于临界值,阳性对照样本检测结果均为刚性.结论 ASPCR是一种快速、灵敏、准确的检测HIV-1微量耐药突变的方法,可为临床抗病毒治疗提供理论指导.     

Non-invasive flow cytometric monitoring of pHi in cell culture processes using EGFP

We have described a new method for monitoring of cell culture processes using green fluorescent protein (GFP) fluorescent intensity. GFP has been used as a non-invasive fluorescent reporter for various cellular processes. In this study, enhanced (EGFP) was found to be a very sensitive indicator of pHi in in vitro cell culture, and responded rapidly to extracellular pH (pHe) changes. EGFP transfected cells were evaluated for pHi changes by flow cytometry, by measuring EGFP fluorescent intensity, and compared to that of the pH-sensitive fluoroprobe, SNARF. EGFP intensity was found to reflect pHi values of cells at different pHe in the presence of nigericin and was affected by the addition of HCl and NaOH. Significant changes in pHi were detected at different stages of batch culture and when using different cell density and media composition. The EGFP assay can be used to minimise the perturbation of cells and processes under study, thus leading to accurate information about the physiological state of single cells in a population. The results establish the application of EGFP as a non-invasive indicator of pHi for monitoring of mammalian cell culture processes.