Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy

Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

“…A 96% overall amplification success of the gp120/gp41 fragment from plasma samples with Ն1,000 copies/ml of HIV RNA, and the use of proprietary universal primers, not only ensured successful amplification of samples of diverse HIV-1 subtypes but also avoided amplification of nonspecific products from endogenous or any of the related viruses tested. Moreover, the subtype B backbone (HIV-1 NL4-3 ) used to clone the patient-derived gp120/gp41 region was compatible not only with env fragments from subtype B wild-type and multidrugresistant strains but also with that from all non-B HIV-1 group M subtypes analyzed (35). Furthermore, the VERITROP assay is efficient and reproducible, with a turnaround time (11 days) comparable to other phenotypic HIV-1 tropism tests, such as the enhanced-sensitivity Trofile assay (16 days) (42), the RVA Toulouse-TTT (9 days) (57), and TROCAI (21 to 42 days) (53).…”

“…Following the identification of the env gene, primarily the V3 region of gp120, as the main determinant for HIV-1 coreceptor tropism, a multitude of cell-based (phenotypic) and sequencingbased (genotypic) approaches have been developed to estimate and quantify the ability of the virus to use any or both receptors to enter the host cell (reviewed in references 12, 14, 15, 21, 23, and 47). Here, we have developed a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on an innovative yeast-based cloning strategy (34,35) and a sensitive cell-to-cell fusion assay (19).…”

“…Thus, a viable alternative has been the use of env recombinant viruses and reporter cell lines expressing HIV-1 receptor and coreceptors to evaluate HIV-1 coreceptor usage (16,18,53), including the only commercially available assay (Trofile; Monogram Biosciences, South San Francisco, CA) (17,42). VERITROP is based on the introduction of patient-derived env fragments into a noninfectious vector using yeast-based cloning (34,35) and a modified version of the ␣-complementation assay for env-mediated cell-to-cell fusion (19). Yeast-based cloning is approximately 100-fold more efficient than bacterium-based restriction enzyme cloning or mammalian cell-based recombination, providing a larger number of unique clones and thereby better representation of the intrapatient HIV-1 population than other cloning methodologies (34).…”

“…The HIV-1 gp120/ gp41-coding region was introduced into a vector using an innovative yeast-based cloning technology (34,35) with minor modifications. Briefly, PCR products spanning the gp120/gp41-coding region of HIV-1 were introduced via yeast homologous recombination into the pRECnfl-LEU-⌬Env(gp120-tatex2)/URA3 vector containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the gp120/gp41 HIV-1 coding sequence (Fig.…”

Novel Two-Round Phenotypic Assay for Protease Inhibitor Susceptibility Testing of Recombinant and Primary HIV-1 Isolates

“…Even so, current standard phenotypic assays do not include the patient-derived full-length Gag region in the recombinant viruses used for PI susceptibility evaluation. Indeed, most commercially available and in-house methods for the determination of PI susceptibility are based on the use of recombinant pseudoviruses or replication-competent chimeric viruses, which harbor the patient-derived 3= half of gag (containing the protein p7 C terminus, p1, and p6) and the whole PR-coding region (6,11,52,54). In order to avoid the drawback of partial cloning of the viral genome, we designed and evaluated a new two-round experimental infection strategy to test the susceptibility of primary viral isolates to PIs.…”