Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.
We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.
The wide scale application of dried blood spots (DBS) as a collection tool for low-cost HIV drug resistance testing requires a greater understanding of the accuracy of DBS for genotype analysis and the stability of DBS under various environmental conditions. Analysis of a 50microl DBS via a single amplicon, nested PCR-based in-house assay (the Burnet genotyping assay) showed an average nucleotide concordance of 98.9% with plasma samples, although only 65% of nucleotide mixtures detected in plasma were also detected within DBS. The analysis of three DBS resulted in the detection of a greater number of nucleotide mixtures (72 and 109 mixtures detected within one and three DBS, respectively, n=10). Two DBS extraction protocols (silica particle; NucliSENS, bioMerieux and spin column extraction; High Pure, Roche) were assessed and found to be equivalent (79% and 84% recovery success respectively, n=19). FTA Elute paper (Whatman) was an inferior DBS collection medium compared to Whatman 903 paper. DBS appeared relatively tolerant to multiple freeze/thaw cycles, with 79% of DBS subjected to ten freeze/thaw cycles successfully amplified compared to 93% of DBS defrosted once (n=14). High temperature (37 degrees C) and high humidity (>90%) substantially impaired DBS recovery within two weeks of storage (38%, n=8), whilst storage at -20 degrees C or 4 degrees C adequately preserved DBS for this period (100% recovery, n=8). Therefore, whilst DBS are suitable for HIV drug resistance surveillance, the use of multiple DBS may be required to ensure accurate detection of minor HIV quasispecies and short-term storage of samples at either 4 degrees C or -20 degrees C is recommended.
We evaluated the new low-cost ExaVir Load (version 3) reverse transcriptase viral load assay against the Roche Cobas Amplicor assay. Results for samples tested using the reverse transcriptase assay correlated well with those obtained with the Roche assay (r ؍ 0.85; n ؍ 202). The version 3 reverse transcriptase assay shows improved sensitivity compared to the previous version.
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