A new approach to grant review assessments: score, then rank.

BACKGROUND: In many grant review settings, proposals are selected for funding on the basis of summary statistics of review ratings. Challenges of this approach (including the presence of ties and unclear ordering of funding preference for proposals) could be mitigated if rankings such as top-k preferences or paired comparisons, which are local evaluations that enforce ordering across proposals, were also collected and incorporated in the analysis of review ratings. However, analyzing ratings and rankings simultaneously has not been done until recently. This paper describes a practical method for integrating rankings and scores and demonstrates its usefulness for making funding decisions in real-world applications. METHODS: We first present the application of our existing joint model for rankings and ratings, the Mallows-Binomial, in obtaining an integrated score for each proposal and generating the induced preference ordering. We then apply this methodology to several theoretical "toy" examples of rating and ranking data, designed to demonstrate specific properties of the model. We then describe an innovative protocol for collecting rankings of the top-six proposals as an add-on to the typical peer review scoring procedures and provide a case study using actual peer review data to exemplify the output and how the model can appropriately resolve judges' evaluations. RESULTS: For the theoretical examples, we show how the model can provide a preference order to equally rated proposals by incorporating rankings, to proposals using ratings and only partial rankings (and how they differ from a ratings-only approach) and to proposals where judges provide internally inconsistent ratings/rankings and outlier scoring. Finally, we discuss how, using real world panel data, this method can provide information about funding priority with a level of accuracy in a well-suited format for research funding decisions. CONCLUSIONS: A methodology is provided to collect and employ both rating and ranking data in peer review assessments of proposal submission quality, highlighting several advantages over methods relying on ratings alone. This method leverages information to most accurately distill reviewer opinion into a useful output to make an informed funding decision and is general enough to be applied to settings such as in the NIH panel review process.

Gender differences in peer reviewed grant applications, awards, and amounts: a systematic review and meta-analysis.

BACKGROUND: Differential participation and success in grant applications may contribute to women's lesser representation in the sciences. This study's objective was to conduct a systematic review and meta-analysis to address the question of gender differences in grant award acceptance rates and reapplication award acceptance rates (potential bias in peer review outcomes) and other grant outcomes. METHODS: The review was registered on PROSPERO (CRD42021232153) and conducted in accordance with PRISMA 2020 standards. We searched Academic Search Complete, PubMed, and Web of Science for the timeframe 1 January 2005 to 31 December 2020, and forward and backward citations. Studies were included that reported data, by gender, on any of the following: grant applications or reapplications, awards, award amounts, award acceptance rates, or reapplication award acceptance rates. Studies that duplicated data reported in another study were excluded. Gender differences were investigated by meta-analyses and generalized linear mixed models. Doi plots and LFK indices were used to assess reporting bias. RESULTS: The searches identified 199 records, of which 13 were eligible. An additional 42 sources from forward and backward searches were eligible, for a total of 55 sources with data on one or more outcomes. The data from these studies ranged from 1975 to 2020: 49 sources were published papers and six were funders' reports (the latter were identified by forwards and backwards searches). Twenty-nine studies reported person-level data, 25 reported application-level data, and one study reported both: person-level data were used in analyses. Award acceptance rates were 1% higher for men, which was not significantly different from women (95% CI 3% more for men to 1% more for women, k = 36, n = 303,795 awards and 1,277,442 applications, I2 = 84%). Reapplication award acceptance rates were significantly higher for men (9%, 95% CI 18% to 1%, k = 7, n = 7319 applications and 3324 awards, I2 = 63%). Women received smaller award amounts (g = -2.28, 95% CI -4.92 to 0.36, k = 13, n = 212,935, I2 = 100%). CONCLUSIONS: The proportions of women that applied for grants, re-applied, accepted awards, and accepted awards after reapplication were less than the proportion of eligible women. However, the award acceptance rate was similar for women and men, implying no gender bias in this peer reviewed grant outcome. Women received smaller awards and fewer awards after re-applying, which may negatively affect continued scientific productivity. Greater transparency is needed to monitor and verify these data globally.

Peer review: Risk and risk tolerance.

Peer review, commonly used in grant funding decisions, relies on scientists' ability to evaluate research proposals' quality. Such judgments are sometimes beyond reviewers' discriminatory power and could lead to a reliance on subjective biases, including preferences for lower risk, incremental projects. However, peer reviewers' risk tolerance has not been well studied. We conducted a cross-sectional experiment of peer reviewers' evaluations of mock primary reviewers' comments in which the level and sources of risks and weaknesses were manipulated. Here we show that proposal risks more strongly predicted reviewers' scores than proposal strengths based on mock proposal evaluations. Risk tolerance was not predictive of scores but reviewer scoring leniency was predictive of overall and criteria scores. The evaluation of risks dominates reviewers' evaluation of research proposals and is a source of inter-reviewer variability. These results suggest that reviewer scoring variability may be attributed to the interpretation of proposal risks, and could benefit from intervention to improve the reliability of reviews. Additionally, the valuation of risk drives proposal evaluations and may reduce the chances that risky, but highly impactful science, is supported.

Grant Review Feedback: Appropriateness and Usefulness.

The primary goal of the peer review of research grant proposals is to evaluate their quality for the funding agency. An important secondary goal is to provide constructive feedback to applicants for their resubmissions. However, little is known about whether review feedback achieves this goal. In this paper, we present a multi-methods analysis of responses from grant applicants regarding their perceptions of the effectiveness and appropriateness of peer review feedback they received from grant submissions. Overall, 56-60% of applicants determined the feedback to be appropriate (fair, well-written, and well-informed), although their judgments were more favorable if their recent application was funded. Importantly, independent of funding success, women found the feedback better written than men, and more white applicants found the feedback to be fair than non-white applicants. Also, perceptions of a variety of biases were specifically reported in respondents' feedback. Less than 40% of applicants found the feedback to be very useful in informing their research and improving grantsmanship and future submissions. Further, negative perceptions of the appropriateness of review feedback were positively correlated with more negative perceptions of feedback usefulness. Importantly, respondents suggested that highly competitive funding pay-lines and poor inter-panel reliability limited the usefulness of review feedback. Overall, these results suggest that more effort is needed to ensure that appropriate and useful feedback is provided to all applicants, bolstering the equity of the review process and likely improving the quality of resubmitted proposals.

Grant reviewer perceptions of the quality, effectiveness, and influence of panel discussion.

BACKGROUND: Funding agencies have long used panel discussion in the peer review of research grant proposals as a way to utilize a set of expertise and perspectives in making funding decisions. Little research has examined the quality of panel discussions and how effectively they are facilitated. METHODS: Here, we present a mixed-method analysis of data from a survey of reviewers focused on their perceptions of the quality, effectiveness, and influence of panel discussion from their last peer review experience. RESULTS: Reviewers indicated that panel discussions were viewed favorably in terms of participation, clarifying differing opinions, informing unassigned reviewers, and chair facilitation. However, some reviewers mentioned issues with panel discussions, including an uneven focus, limited participation from unassigned reviewers, and short discussion times. Most reviewers felt the discussions affected the review outcome, helped in choosing the best science, and were generally fair and balanced. However, those who felt the discussion did not affect the outcome were also more likely to evaluate panel communication negatively, and several reviewers mentioned potential sources of bias related to the discussion. While respondents strongly acknowledged the importance of the chair in ensuring appropriate facilitation of the discussion to influence scoring and to limit the influence of potential sources of bias from the discussion on scoring, nearly a third of respondents did not find the chair of their most recent panel to have performed these roles effectively. CONCLUSIONS: It is likely that improving chair training in the management of discussion as well as creating review procedures that are informed by the science of leadership and team communication would improve review processes and proposal review reliability.

The Participation and Motivations of Grant Peer Reviewers: A Comprehensive Survey.

Scientific peer reviewers play an integral role in the grant selection process, yet very little has been reported on the levels of participation or the motivations of scientists to take part in peer review. The American Institute of Biological Sciences (AIBS) developed a comprehensive peer review survey that examined the motivations and levels of participation of grant reviewers. The survey was disseminated to 13,091 scientists in AIBS's proprietary database. Of the 874 respondents, 76% indicated they had reviewed grant applications in the last 3 years; however, the number of reviews was unevenly distributed across this sample. Higher review loads were associated with respondents who had submitted more grant proposals over this time period, some of whom were likely to be study section members for large funding agencies. The most prevalent reason to participate in a review was to give back to the scientific community (especially among frequent grant submitters) and the most common reason to decline an invitation to review was lack of time. Interestingly, few suggested that expectation from the funding agency was a motivation to review. Most felt that review participation positively influenced their careers through improving grantsmanship and exposure to new scientific ideas. Of those who reviewed, respondents reported dedicating 2-5% of their total annual work time to grant review and, based on their self-reported maximum review loads, it is estimated they are participating at 56-87% of their capacity, which may have important implications regarding the sustainability of the system. Overall, it is clear that participation in peer review is uneven and in some cases near capacity, and more needs to be done to create new motivations and incentives to increase the future pool of reviewers.

The Influence of Peer Reviewer Expertise on the Evaluation of Research Funding Applications.

Although the scientific peer review process is crucial to distributing research investments, little has been reported about the decision-making processes used by reviewers. One key attribute likely to be important for decision-making is reviewer expertise. Recent data from an experimental blinded review utilizing a direct measure of expertise has found that closer intellectual distances between applicant and reviewer lead to harsher evaluations, possibly suggesting that information is differentially sampled across subject-matter expertise levels and across information type (e.g. strengths or weaknesses). However, social and professional networks have been suggested to play a role in reviewer scoring. In an effort to test whether this result can be replicated in a real-world unblinded study utilizing self-assessed reviewer expertise, we conducted a retrospective multi-level regression analysis of 1,450 individual unblinded evaluations of 725 biomedical research funding applications by 1,044 reviewers. Despite the large variability in the scoring data, the results are largely confirmatory of work from blinded reviews, by which a linear relationship between reviewer expertise and their evaluations was observed-reviewers with higher levels of self-assessed expertise tended to be harsher in their evaluations. However, we also found that reviewer and applicant seniority could influence this relationship, suggesting social networks could have subtle influences on reviewer scoring. Overall, these results highlight the need to explore how reviewers utilize their expertise to gather and weight information from the application in making their evaluations.

Frequency and Type of Conflicts of Interest in the Peer Review of Basic Biomedical Research Funding Applications: Self-Reporting Versus Manual Detection.

Despite the presumed frequency of conflicts of interest in scientific peer review, there is a paucity of data in the literature reporting on the frequency and type of conflicts that occur, particularly with regard to the peer review of basic science applications. To address this gap, the American Institute of Biological Sciences (AIBS) conducted a retrospective analysis of conflict of interest data from the peer review of 282 biomedical research applications via several onsite review panels. The overall conflicted-ness of these panels was significantly lower than that reported for regulatory review. In addition, the majority of identified conflicts were institutional or collaborative in nature. No direct financial conflicts were identified, although this is likely due to the relatively basic science nature of the research. It was also found that 65 % of identified conflicts were manually detected by AIBS staff searching reviewer CVs and application documents, with the remaining 35 % resulting from self-reporting. The lack of self-reporting may be in part attributed to a lack of perceived risk of the conflict. This result indicates that many potential conflicts go unreported in peer review, underscoring the importance of improving detection methods and standardizing the reporting of reviewer and applicant conflict of interest information.

A retrospective analysis of the effect of discussion in teleconference and face-to-face scientific peer-review panels.

OBJECTIVE: With the use of teleconferencing for grant peer-review panels increasing, further studies are necessary to determine the efficacy of the teleconference setting compared to the traditional onsite/face-to-face setting. The objective of this analysis was to examine the effects of discussion, namely changes in application scoring premeeting and postdiscussion, in these settings. We also investigated other parameters, including the magnitude of score shifts and application discussion time in face-to-face and teleconference review settings. DESIGN: The investigation involved a retrospective, quantitative analysis of premeeting and postdiscussion scores and discussion times for teleconference and face-to-face review panels. The analysis included 260 and 212 application score data points and 212 and 171 discussion time data points for the face-to-face and teleconference settings, respectively. RESULTS: The effect of discussion was found to be small, on average, in both settings. However, discussion was found to be important for at least 10% of applications, regardless of setting, with these applications moving over a potential funding line in either direction (fundable to unfundable or vice versa). Small differences were uncovered relating to the effect of discussion between settings, including a decrease in the magnitude of the effect in the teleconference panels as compared to face-to-face. Discussion time (despite teleconferences having shorter discussions) was observed to have little influence on the magnitude of the effect of discussion. Additionally, panel discussion was found to often result in a poorer score (as opposed to an improvement) when compared to reviewer premeeting scores. This was true regardless of setting or assigned reviewer type (primary or secondary reviewer). CONCLUSIONS: Subtle differences were observed between settings, potentially due to reduced engagement in teleconferences. Overall, further research is required on the psychology of decision-making, team performance and persuasion to better elucidate the group dynamics of telephonic and virtual ad-hoc peer-review panels.

The validation of peer review through research impact measures and the implications for funding strategies.

There is a paucity of data in the literature concerning the validation of the grant application peer review process, which is used to help direct billions of dollars in research funds. Ultimately, this validation will hinge upon empirical data relating the output of funded projects to the predictions implicit in the overall scientific merit scores from the peer review of submitted applications. In an effort to address this need, the American Institute of Biological Sciences (AIBS) conducted a retrospective analysis of peer review data of 2,063 applications submitted to a particular research program and the bibliometric output of the resultant 227 funded projects over an 8-year period. Peer review scores associated with applications were found to be moderately correlated with the total time-adjusted citation output of funded projects, although a high degree of variability existed in the data. Analysis over time revealed that as average annual scores of all applications (both funded and unfunded) submitted to this program improved with time, the average annual citation output per application increased. Citation impact did not correlate with the amount of funds awarded per application or with the total annual programmatic budget. However, the number of funded applications per year was found to correlate well with total annual citation impact, suggesting that improving funding success rates by reducing the size of awards may be an efficient strategy to optimize the scientific impact of research program portfolios. This strategy must be weighed against the need for a balanced research portfolio and the inherent high costs of some areas of research. The relationship observed between peer review scores and bibliometric output lays the groundwork for establishing a model system for future prospective testing of the validity of peer review formats and procedures.

Teleconference versus face-to-face scientific peer review of grant application: effects on review outcomes.

Teleconferencing as a setting for scientific peer review is an attractive option for funding agencies, given the substantial environmental and cost savings. Despite this, there is a paucity of published data validating teleconference-based peer review compared to the face-to-face process. Our aim was to conduct a retrospective analysis of scientific peer review data to investigate whether review setting has an effect on review process and outcome measures. We analyzed reviewer scoring data from a research program that had recently modified the review setting from face-to-face to a teleconference format with minimal changes to the overall review procedures. This analysis included approximately 1600 applications over a 4-year period: two years of face-to-face panel meetings compared to two years of teleconference meetings. The average overall scientific merit scores, score distribution, standard deviations and reviewer inter-rater reliability statistics were measured, as well as reviewer demographics and length of time discussing applications. The data indicate that few differences are evident between face-to-face and teleconference settings with regard to average overall scientific merit score, scoring distribution, standard deviation, reviewer demographics or inter-rater reliability. However, some difference was found in the discussion time. These findings suggest that most review outcome measures are unaffected by review setting, which would support the trend of using teleconference reviews rather than face-to-face meetings. However, further studies are needed to assess any correlations among discussion time, application funding and the productivity of funded research projects.

Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion.

BACKGROUND: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4. RESULTS: HIV-1 and HIV-2 Env-mediated cell fusion occurred with half times of about 60 and 30 min, respectively. Binding experiments of soluble HIV gp120 proteins to CD4 and co-receptor did not correlate with the differences in kinetics of fusion mediated by the three different HIV Envs. However, escape from inhibition by reagents that block gp120-CD4 binding, CD4-induced CXCR4 binding and 6-helix bundle formation, respectively, indicated large difference between HIV-1 and HIV-2 envelope glycoproteins in their CD4-induced rates of engagement with CXCR4. CONCLUSION: The HIV-2 Env proteins studied here exhibited a significantly reduced window of time between the engagement of gp120 with CD4 and exposure of the CXCR4 binding site on gp120 as compared with HIV-1IIIB Env. The efficiency with which HIV-2 Env undergoes this CD4-induced conformational change is the major cause of the relatively rapid rate of HIV-2 Env mediated-fusion.

Sphingolipids, cholesterol, and HIV-1: a paradigm in viral fusion.

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37 degrees C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.

Theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation.

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Elevated expression of GM3 in receptor-bearing targets confers resistance to human immunodeficiency virus type 1 fusion.

GM3, a major ganglioside of T lymphocytes, promotes human immunodeficiency virus type 1 (HIV-1) entry via interactions with HIV-1 receptors and the viral envelope glycoprotein (Env). Increased GM3 levels in T lymphocytes and the appearance of anti-GM3 antibodies in AIDS patients have been reported earlier. In this study, we investigated the effect of GM3 regulation on HIV-1 entry by utilizing a mouse cell line (B16F10), which expresses exceptionally high levels of GM3. Strikingly, B16 cells bearing CD4, CXCR4, and/or CCR5 were highly resistant to CD4-dependent HIV-1 Env-mediated membrane fusion. In contrast, these targets supported membrane fusion mediated by CD4-requiring HIV-2, SIV, and CD4-independent HIV-1 Envs. Coreceptor function was not impaired by GM3 overexpression as indicated by Ca(2+) fluxes mediated by the CXCR4 ligand SDF-1alpha and the CCR5 ligand MIP-1beta. Reduction in GM3 levels of B16 target cells resulted in a significant recovery of CD4-dependent HIV-1 Env-mediated fusion. We propose that GM3 in the plasma membrane blocks HIV-1 Env-mediated fusion by interfering with the lateral association of HIV-1 receptors. Our findings offer a novel mechanism of interplay between membrane lipids and receptors by which host cells may escape viral infections.

The stability of the intact envelope glycoproteins is a major determinant of sensitivity of HIV/SIV to peptidic fusion inhibitors.

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC(50)<10nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC(50)>100nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.

Temperature-dependent intermediates in HIV-1 envelope glycoprotein-mediated fusion revealed by inhibitors that target N- and C-terminal helical regions of HIV-1 gp41.

Peptides derived from the N- (N-HR) and C- (C-HR) terminal heptad repeat regions adjacent to the fusion peptide and transmembrane domains, respectively, of human immunodeficiency virus (HIV)-1 gp41 inhibit HIV-1 viral envelope glycoproteins (Env)-mediated cell fusion specifically. The mechanism of HIV-1 Env-mediated cell fusion and its inhibition by agents that target the N- and C-HR regions was investigated. Priming experiments with Env-expressing cells indicate that the N-HR region but not the C-HR region is exposed by treatment with sCD4 at 31 degrees C, whereas both the N- and C-HR regions are exposed at 37 degrees C.

Functional expression of CD4, CXCR4, and CCR5 in glycosphingolipid-deficient mouse melanoma GM95 cells and susceptibility to HIV-1 envelope glycoprotein-triggered membrane fusion.

We had previously reported that glycosphingolipids (GSL) support human immunodeficiency virus type 1 (HIV-1) entry. In this study, we further examined this issue by expressing HIV-1 receptors in GSL-deficient GM95 cells. GM95 cells expressing low levels of CD4 and CXCR4 or CCR5 did not support HIV-1 Env-mediated fusion. However, higher expression of these receptors rendered GM95 cells highly susceptible to fusion with cells expressing appropriate HIV-1 envelope glycoproteins (HIV-1 Envs). The GM95 cells exhibited a different fusion phenotype when compared with GSL(+) NIH3T3 cells bearing similar receptor levels. Fusion of GM95 targets expressing higher levels of CD4 and coreceptors occurred at 25 degrees C and was sensitive to cholesterol depletion or disruption of the cytoskeleton. In contrast, the fusion threshold of NIH3T3CD4X4/R5 targets was at >/=28 degrees C as previously reported and was insensitive to cholesterol depletion or cytoskeletal network disruption. On the basis of these observations, we propose that target membrane GSLs support HIV-1 Env-mediated fusion at low density of receptors by stabilizing receptor pools in natural targets.

The C- and the N-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively.

To infect target cells, HIV-1 employs a virally encoded transmembrane protein (gp41) to fuse its viral envelope with the target cell plasma membrane. We describe the gp41 ectodomain as comprised of N- and C-terminal subdomains, each containing a heptad repeat as well as a fusogenic region, whose organization is mirrored by the intervening loop region. Recent evidence indicates that the gp41 directed fusion reaction proceeds to initial pore formation prior to gp41 folding into its low energy hairpin conformation. This implies that exposed regions of the gp41 ectodomain are responsible for the bulk of the fusion work, probably through direct protein-membrane interactions. Prevalent fusion models contend that the gp41 ectodomain initially interacts with the target cell surface through its highly hydrophobic N terminus, which is believed to insert into the target membrane, thereby linking the virus to the target cell. This arrangement allows the N-terminal subdomain to interact with the target cell surface, whereas the C-terminal subdomain remains proximal to the virion, allowing interaction with the viral envelope. The composition of the viral envelope and the target cell surface differ due to the virus budding from raft microdomains. We show here that constructs corresponding to the C-terminal subdomain specifically destabilize ordered and cholesterol rich membranes (33 molar %), whereas the N-terminal subdomain is more effective in fusing both unordered cholesterol-free membranes and those containing lower amounts of cholesterol (10 molar %). Moreover we show that, in the context of the C-terminal subdomain, the heptad repeat contributes helical structure, which may describe the enhanced inhibitory effect of the C-terminal subdomain relative to the C-terminal heptad repeat (C34) alone. Our results are discussed in light of recent findings that showcase the role of exposed gp41 regions in effecting membrane fusion.

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review).

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.