Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

Development and performance of conventional HIV-1 phenotyping (Antivirogram®) and genotype-based calculated phenotyping assay (virco®TYPE HIV-1) on protease and reverse transcriptase genes to evaluate drug resistance.

OBJECTIVES: A wide array of monitoring tests is commercially available to gauge HIV-1 disease progression and the overall health status of an HIV-1-infected patient. Viral load tests provide a picture of viral activity, while CD4 cell counts shed light on the immune status and can help physicians to prevent the development of opportunistic infections in patients. On the other hand, genotypic and phenotypic resistance testing and therapeutic drug monitoring help to optimize HIV-1 antiretroviral therapy. Resistance testing is currently recommended within the standard of care guidelines to aid the choice of new drug regimens following treatment failure(s). METHODS: Genotypic testing described here is based on the amplification and sequencing of an HIV-1 protease (PR) and reverse transcriptase (RT) region from a patient sample to identify resistance mutations associated with PR and RT inhibitor resistance. A genotypic test takes a week to perform and the results are reported as a list of detected mutations. The virco®TYPE HIV-1 report uses genotypic data to predict phenotypic susceptibility by linear regression modeling that uses a large correlative database of genotype-phenotype pairs. Phenotypic testing measures the ability of the virus to replicate in the presence of a drug and provides a direct measurement of drug susceptibility in vitro. Since phenotypic analysis is laborious and time consuming (28 days), genotypic resistance testing is currently the standard reference method used for HIV-1 resistance testing. However, a phenotypic test is important when a patient harbors virus with complex genetic patterns, or when the mutational resistance profile for a particular drug is not well-characterized. RESULTS AND CONCLUSIONS: Some of the currently used resistance tests are partially automated enabling laboratories to increase overall efficiency. However, maximum automation and standardization of the process, instruments and software that we have described here can overcome many of the problems encountered with current tests and aims at having a compliant, high-throughput, diagnostic laboratory, which can guarantee sample integrity from sample reception to result reporting. We also describe in detail the development and performance of virco®TYPE HIV-1 (genotype) and Antivirogram® (phenotype) assay on PR and RT genes to evaluate antiretroviral resistance.

A synthetic HIV-1 subtype C backbone generates comparable PR and RT resistance profiles to a subtype B backbone in a recombinant virus assay.

In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment.

BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

HIV-1 V3 envelope deep sequencing for clinical plasma specimens failing in phenotypic tropism assays.

BACKGROUND: HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain. RESULTS: A cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly. CONCLUSIONS: Although only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.

Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity.

OBJECTIVES: The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. METHODS: Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. RESULTS: Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. CONCLUSIONS: We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

Secondary integrase resistance mutations found in HIV-1 minority quasispecies in integrase therapy-naive patients have little or no effect on susceptibility to integrase inhibitors.

The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens.

BACKGROUND: Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS: We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS: Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS: Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes.

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1.

In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Clade-specific HIV-1 integrase polymorphisms do not reduce raltegravir and elvitegravir phenotypic susceptibility.

The contribution of clade-specific polymorphisms in the HIV-1 integrase gene towards integrase inhibitor phenotypic susceptibility was tested on 137 clinical isolates, of which 60 were non-clade B strains. Control Q148R mutant virus showed fold change values of 17.85 +/- 2.77 and 88.94 +/- 9.02 for raltegravir and elvitegravir, respectively, whereas the average fold change for the clinical samples was 0.91 +/- 0.40, and 0.84 +/- 0.37. Phenotypic testing proved that clade-specific integrase polymorphisms do not contribute to reduced susceptibility towards integrase inhibitors.

Performance of a population-based HIV-1 tropism phenotypic assay and correlation with V3 genotypic prediction tools in recent HIV-1 seroconverters.

BACKGROUND: Pure X4 and X4R5 dual-tropic viruses may be recognized in approximately 15% of drug-naive HIV-1-positive patients. CCR5 antagonists are active against R5 viruses; therefore, HIV tropism should be known before their prescription. PATIENTS AND METHODS: A population-based phenotypic assay was performed in 61 recent HIV-1 seroconverters. The results were compared with those obtained using 8 different predictor software programs (C4.5, C4.5 with 8 and 12, PART, SVM, Charge Rule, PSSMsinsi, PSSMx4r5, and geno2pheno), which are freely available at 3 different Web sites and use V3 sequences derived from patient's viruses. RESULTS: Phenotypic testing reported X4R5 dual-tropic viruses in 10 (16.4%) patients. CD4 cell counts and viral loads were significantly lower in X4R5 dual-tropic (450 cells/microL and 3.9 log HIV RNA copies/mL) than in R5 viruses (629 cells/microL, 4.5 log HIV RNA copies/mL) (P<0.05). The overall concordance of genotype and phenotype was relatively good (>80%). Although specificity was >90% using all but 1 genotypic predictor (geno2pheno), however, the sensitivity for the detection of X4 variants was low (<30%), except for SVM and geno2pheno (70%). CONCLUSIONS: The prevalence of X4 and X4/R5 dual-tropic viruses in recent HIV seroconverters is 16%. Current genotypic algorithms need to be improved for the estimation of HIV-1 coreceptor use before moving to the clinic. This information is crucial for the selection of candidates to receive CCR5 antagonists in places where phenotypic tropism assays may not be feasible.

HIV-1 coreceptor usage determination in clinical isolates using clonal and population-based genotypic and phenotypic assays.

Orally bioavailable CXCR4 and CCR5 coreceptor antagonists are being developed for the treatment of HIV-1 infection. A new tropism-testing platform, which offers various options depending on the needs, was established. Each option has specific characteristics in terms of sensitivity, information, throughput and cost. The platform consists of four assays, all based on a one-step RT-PCR of the main part of the HIV envelope glycoprotein gp120 (called 'NH(2)-V4'). Population-based sequencing of gp120's V3 loop is generally cheap and easy to run, and was chosen as the first test in the platform's cascade. Given its drawbacks such as limited sensitivity, additional tests were developed. A sensitive assay using NH(2)-V4 gp120 clonal sequencing and tropism prediction enabled us to demonstrate the quasispecies diversity present in 13 patient samples. For phenotyping, an eGFP-containing HIV backbone deleted for NH(2)-V4 was constructed and used for clonal and population tropism determination. As expected, clonal NH(2)-V4 gp120 phenotyping demonstrated significant correlation between prediction algorithms and phenotype-based classification. The absence of the N-linked glycosylation motif in V3 was associated with CXCR4 usage. Finally, population NH(2)-V4 gp120 phenotypic tropism determination appeared to be a promising tool for the detection of minority species present in the amplified envelope fragments.

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions.

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca(2+), demonstrated its Mn(2+)-transporting activity, showed its Ca(2+)- and Mn(2+)-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of (45)Ca(2+) (K(0.5) = 0.27 microm) or (54)Mn(2+) transport into an A23187-releasable compartment.

The Ca2+/Mn2+ pumps in the Golgi apparatus.

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.

SPCA1 pumps and Hailey-Hailey disease.

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated.

Hailey-Hailey disease: identification of novel mutations in ATP2C1 and effect of missense mutation A528P on protein expression levels.

ATP2C1, encoding the human secretory pathway Ca(2+)-ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey disease (HHD), an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis. In this study, we used denaturing high-performance liquid chromatography to screen all 28 exons and flanking intron boundaries of ATP2C1 for mutations in 9 HHD patients. Nine different mutations were identified. Five of these mutations, including one nonsense, one deletion, two splice-site, and one missense mutation, have not been previously reported. Recently, functional analysis of a series of site-specific mutants, designed to mimic missense mutations found in ATP2C1, uncovered specific defects in Ca(2+) and/or Mn(2+) transport and protein expression in mutant hSPCA1 polypeptides. In order to investigate the molecular and physiological basis of HHD in the patient carrying missense mutation A528P, located in the putative nucleotide binding domain of the molecule, site-directed mutagenesis was employed to introduce this mutation into the wild-type ATP2C1 (hSPCA1) sequence. Functional analyses of HHD-mutant A528P demonstrated a low level of protein expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. Analogous to conclusions drawn from our previous studies, these results further support the theory of haploinsufficiency as a prevalent mechanism for the dominant inheritance of HHD, by suggesting that the level of hSPCA1 in epidermal cells is critical.

The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference.

The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses.

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.