Kenneth R. Henry

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates

Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinetics, or to develop personalized vaccines. Extreme HIV-1 heterogeneity leaves few restriction enzyme sites for bacterial cloning strategies. In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus species such as hepatitis C virus and influenza virus.

The Actin-Regulating Kinase Prk1p Negatively Regulates Scd5p, a Suppressor of Clathrin Deficiency, in Actin Organization and Endocytosis

Endocytosis is a dynamic process requiring a network of interacting proteins that assemble and disassemble during cargo capture and vesicle formation. A major mechanism for regulation of this process involves the reversible phosphorylation of endocytic factors. Recently, members of a new kinase family, the Ark/Prk kinases, which include mammalian AAK1 and GAK as well as yeast Prk1p, Ark1p, and Akl1p, were shown to regulate components of the endocytic machinery. These include animal AP-1/AP-2 mu chains and yeast Pan1p (Eps15-like), Sla1p, and epsins, but other potential targets are likely. SCD5, an essential yeast gene, was identified as a suppressor of clathrin deficiency. We also showed that Scd5p is required for normal cortical actin organization and endocytosis, possibly as a targeting subunit for protein phosphatase type 1 (PP1). Scd5p contains a central triple repeat (3R) motif related to a known Prk1p consensus phosphorylation site L/IxxQxTG, except that Q is replaced by T. In this study we demonstrate that the Scd5p 3R sequence is phosphorylated by Prk1p to negatively regulate Scd5p. Furthermore, we show that Prk1p, Ark1p, and Akl1p have different substrate specificities and play distinct roles in actin organization and endocytosis.

Protein phosphatase-1 binding to Scd5p is important for regulation of actin organization and endocytosis in yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the −1 or −2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240–243) and KKVRF (amino acids 272–276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.

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

ABSTRACT CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

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

ABSTRACT Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.

SSLPs to map genetic differences between the 129 inbred strains and closed-colony, random-bred CD-1 mice

Gene targeting is an important tool for analyzing gene function in vivo as well as a means for creating animal models for specific human diseases. Additionally, numerous targeted loci show phenotypes that depend on genetic background, thus providing an even broader understanding of gene function (Threadgill et al. 1995; Sibilia and Wagner 1995; B aribault et al. 1994; Rozmahel et al. 1996). Classical backcross breeding schemes for congenic strain production, which usually takes years, are used to transfer targeted alleles from one inbred strain onto other inbred backgrounds. With the discovery of the highly polymorphic and simple genotyping protocols of SSLPs (simple sequence length polymorphisms; Hamada et al. 1982; Love et al. 1990), marker selection can be used to dramatically reduce the number of generations needed to produce a congenic strain. These markers are also very useful because their high level of polymorphism can be exploited to map the locations of secondary genetic loci involved in genetic background-dependent phenotypic differences. Unfortunately, the 129 strain was not included in the original screen for SSLP allele sizes among a panel of inbred strains (Dietrich et al. 1992). Matouk and associates tabulated allele sizes for 129 mice at numerous SSLP loci (Matouk et al. 1996). However, they used the 129/SvJ substrain, which is actually a recombinant congenic strain, 129cX/Sv (Threadgill et al. I997), as the source for 129 DNA. Many lines carrying targeted mutations are also mated to random-bred mouse stocks such as CD1 because of their high fecundity and excellent matemal characteristics. In order to identify a set of markers that can be used for marker selection during production of congenic strains or for mapping the genetic locations of phenotypic modifiers, we have typed the 129/Sv-+ ryr +P and CD-1 strains at numerous SSLPs. Crude tail DNA was prepared as previously reported (McMahon and Bradley 1990) from a 129/Sv-+ ryr +P and at least three independent CD-1 animals from a closed, random-breeding colony (Charles Rivers, Portage, MI). Approximately 100 ng of each sample was tested in a 20-pA hot start PCR reaction with 40 cycles of 95~ 30 s; 60~ 30 s, after which 10 p~l of each PCR reaction was electrophoresed on 15 cm vertical 8% nondenaturing polyacrylamide gels (R. Shadel, San Francisco, CA), stained with ethidium bromide, and photographed. A 25-bp ladder (BRL, Gaithersburg, MD) was used as a size standard, and allele sizes were estimated on the basis of the known allele sizes in various inbred strains (Dietrich et al. 1996). The MIT (Ob X CAST) F 2 intercross was used for the genetic map locations of the SSLPs (Dietrich et al. 1996; http://www-genome.wi.mit.edu/cgi-bin/ mouse/index). We screened 784 SSLPs to identify markers that differ be-

Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA.

Background The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. Methodology/Principal Findings We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. Conclusions/Significance We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation.

Viral fitness: relation to drug resistance mutations and mechanisms involved: nucleoside reverse transcriptase inhibitor mutations.

Purpose of reviewNucleoside and nucleotide reverse transcriptase inhibitors constitute the backbone of highly active antiretroviral therapy in the treatment of HIV-1 infection. One of the major obstacles in achieving the long-term efficacy of anti-HIV-1 therapy is the development of resistance. The advent of resistance mutations is usually accompanied by a change in viral replicative fitness. This review focuses on the most common nucleoside reverse transcriptase inhibitor-associated mutations and their effects on HIV-1 replicative fitness. Recent findingsRecent studies have explained the two main mechanisms of resistance to nucleoside reverse transcriptase inhibitors and their role in HIV-1 replicative fitness. The first involves mutations directly interfering with binding or incorporation and seems to impact replicative fitness more adversely than the second mechanism, which involves enhanced excision of the newly incorporated analogue. Further studies have helped explain the antagonistic effects between amino acid substitutions, K65R, L74V, M184V, and thymidine analogue mutations, showing how viral replicative fitness influences the evolution of thymidine analogue resistance pathways. SummaryNucleoside reverse transcriptase inhibitor resistance mutations impact HIV-1 replicative fitness to a lesser extent than protease resistance mutations. The monitoring of viral replicative fitness may help in the management of HIV-1 infection in highly antiretroviral-experienced individuals.

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections

BackgroundNew simian–human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques.ResultsEven though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env’s in the pool, a feature also observed in the HIV establishing new infections in humans.ConclusionDespite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.