Template-constrained cyclic sulfopeptide HIV-1 entry inhibitors.

Template-constrained cyclic sulfopeptides that inhibit HIV-1 entry were rationally designed based on a loop from monoclonal antibody (mAb) 412d. A focused set of sulfopeptides was synthesized using Fmoc-Tyr(SO3DCV)-OH (DCV = 2,2-dichlorovinyl). Three cyclic sulfopeptides that inhibit entry of HIV-1 and complement the activity of known CCR5 antagonists were identified.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

Adaptive mutations in a human immunodeficiency virus type 1 envelope protein with a truncated V3 loop restore function by improving interactions with CD4.

We previously reported that a human immunodeficiency virus type 1 (HIV-1) clade B envelope protein with a severely truncated V3 loop regained function after passage in tissue culture. The adapted virus, termed TA1, retained the V3 truncation, was exquisitely sensitive to neutralization by the CD4 binding site monoclonal antibody b12 and by HIV-positive human sera, used CCR5 to enter cells, and was completely resistant to small molecule CCR5 antagonists. To examine the mechanistic basis for these properties, we singly and in combination introduced each of the 5 mutations from the adapted clone TA1 into the unadapted envelope. We found that single amino acid changes in the C3 region, the V3 loop, and in the fusion peptide were responsible for imparting near-normal levels of envelope function to TA1. T342A, which resulted in the loss of a highly conserved glycosylation site in C3, played the primary role. The adaptive amino acid changes had no impact on CCR5 antagonist resistance but made virus more sensitive to neutralization by antibodies to the CD4 binding site, modestly enhanced affinity for CD4, and made TA1 more responsive to CD4 binding. Specifically, TA1 was triggered by soluble CD4 more readily than the parental Env and, unlike the parental Env, could mediate entry on cells that express low levels of CD4. In contrast, TA1 interacted with CCR5 less efficiently and was highly sensitive to antibodies that bind to the CCR5 N terminus and ECL2. Therefore, enhanced utilization of CD4 is one mechanism by which HIV-1 can overcome mutations in the V3 region that negatively affect CCR5 interactions.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies.

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.

Replicative fidelity of lentiviral vectors produced by transient transfection.

Previous investigations have estimated the human immunodeficiency virus type 1 (HIV) base pair substitution rate to be approximately 10(-4) to 10(-5) per round of viral replication, and HIV has been hypothesized to be more error-prone than other retroviruses. Using a single cycle reversion assay, we unexpectedly found that the reversion rates of HIV, avian leukosis virus and Moloney murine leukemia virus were the same, within statistical error. Because both the viral enzyme reverse transcriptase (RT) and cellular RNA polymerase II (RNAP) are required for viral replication, we hypothesized that the similar reversion rates actually reflect the intrinsic error rate of RNAP, which is the enzyme common to all three retroviruses in the reversion assay. To address this possibility, HIV vectors with the U3 region replaced by a reporter reversion cassette were constructed and vector supernatant produced by transient transfection. All single integrant revertant cell lines showed the identical mutations at both long terminal repeats. This indicates that either RNAP or another cellular enzyme is responsible for these reversions, or that HIV RT only makes errors during first strand synthesis. Additionally, when HIV particles were rescued from an integrated vector as opposed to being produced by transient transfection, the reversion rate was significantly lower, suggesting that one or more factors in the virus-producing cells plays a role in the fidelity of retroviral replication. These results have implications regarding the fidelity of the transgene after transient transfection production of lentiviral vector supernatants.