s2fieldsofstudy: Biology Psychology Medicine

text:
Intermediate Phenotypes Identify Divergent Pathways to Alzheimer's Disease

Background Recent genetic studies have identified a growing number of loci with suggestive evidence of association with susceptibility to Alzheimer's disease (AD). However, little is known of the role of these candidate genes in influencing intermediate phenotypes associated with a diagnosis of AD, including cognitive decline or AD neuropathologic burden. Methods/Principal Findings Thirty-two single nucleotide polymorphisms (SNPs) previously implicated in AD susceptibility were genotyped in 414 subjects with both annual clinical evaluation and completed brain autopsies from the Religious Orders Study and the Rush Memory and Aging Project. Regression analyses evaluated the relation of SNP genotypes to continuous measures of AD neuropathology and cognitive function proximate to death. A SNP in the zinc finger protein 224 gene (ZNF224, rs3746319) was associated with both global AD neuropathology (p = 0.009) and global cognition (p = 0.002); whereas, a SNP at the phosphoenolpyruvate carboxykinase locus (PCK1, rs8192708) was selectively associated with global cognition (p = 3.57×10−4). The association of ZNF224 with cognitive impairment was mediated by neurofibrillary tangles, whereas PCK1 largely influenced cognition independent of AD pathology, as well as Lewy bodies and infarcts. Conclusions/Significance The findings support the association of several loci with AD, and suggest how intermediate phenotypes can enhance analysis of susceptibility loci in this complex genetic disorder.

Introduction
Alzheimer's disease (AD), the most common cause of dementia, leads to progressive loss of memory and other cognitive domains, and is characterized pathologically by the accumulation of extracelluar amyloid plaques and intracellular neurofibrillary tangles. AD likely develops from an interaction of numerous genes along with environmental risk factors, each with modest and incompletely penetrant effects. Linkage studies have identified rare gene mutations as causal in familial, early age-of-onset AD, but these Mendelian variants only explain a small fraction of disease burden in the general population [1]. The identification of susceptibility loci for sporadic, late age-of-onset AD has been more challenging, with numerous reports of candidate gene associations, most of which have not been consistently replicated in follow-up studies [2][3][4]. One notable exception is the apolipoprotein E locus (APOE): the e4 allele is common, increases AD susceptibility 3-fold, and is estimated to explain at least 10% of the population-attributable risk of disease [1]. In addition, the APOE e2 allele is a validated AD protective allele though it is less common, and its effect size is more modest than e4.
Genome-wide association (GWA) studies have emerged as a promising approach to identify susceptibility loci in common diseases with complex genetic inheritance, but until recently, most GWA scans in AD have been relatively underpowered, and identified loci have not been consistently replicated [5][6][7][8][9][10][11][12][13][14]. Increasing sample size is one approach for boosting statistical power, and this strategy has recently led to the identification of several promising new AD susceptibility loci, including CR1, CLU, and PICALM [15,16]. However, clinical heterogeneity remains a significant confounder of the case/control study design in AD, due to the likely inclusion of dementia cases with multiple pathologies, such as cerebrovascular disease or other neurodegenerative conditions. In addition, since AD develops following a protracted pre-clinical phase consisting of mild symptoms, control groups are susceptible to contamination by latent disease cases. Substantial AD pathology is often present in advanced age, including in those with minimal or no cognitive impairment at death [17]. Subjects with significant pathology but subclinical disease are likely to dilute power in an AD case/control association analysis.
One approach to overcoming these obstacles is to study quantitative intermediate phenotypes. The manifestation of the AD clinical syndrome is the final culmination of a sequence of events beginning with genetic and environmental risk factors that trigger intermediate pathological changes, synapse loss and cell death, and ultimately cognitive decline and dementia. Outcome measures selected more proximally along this causal chain are expected be less confounded and more strongly associated with susceptibility loci. In addition, compared to the dichotomous clinical diagnosis, quantitative intermediate phenotypes can capture more of the underlying heritable trait variation, further enhancing statistical power. Based on this promise, a number of studies have begun to take advantage of intermediate phenotypes for genetic association analysis in AD, including neuropsychiatric test measures [18], MRI imaging data [19,20], biomarkers from blood and CSF [21,22], and direct measurements of AD pathology [23]. The latter approach requires access to large study populations with detailed clinical and neuropathologic characterization. The Religious Orders Study and Rush Memory and Aging Project are prospectively following more than 2,300 older persons, all of whom have agreed to annual clinical evaluation and brain donation at death. More than 800 autopsies have been completed to date, and quantitative analyses of amyloid and tangle burden has been performed on nearly 600. In a recent study of APOE in this cohort, we found that intermediate cognitive and pathological phenotypes substantially increase power for genetic association analysis [23]. In addition, using neuropathologic phenotypes, the association between APOE and cognitive impairment was previously shown to be mediated by a sequential cascade of amyloid plaque formation and subsequent development of neurofibrillary tangle pathology [24,25]. Therefore, beyond enhancing power for association analysis, intermediate phenotypes hold the additional promise of testing mechanistic hypotheses of gene action.
In this study, we extend our previous work to evaluate several candidate AD susceptibility loci for associations with intermediate phenotypes relevant to AD. Thirty-two candidate SNPs were selected based on their discovery in AD GWA studies and/or evidence from the AlzGene online meta-analyses [2,26]. SNPs were genotyped in more than 400 subjects with detailed cognitive and pathological data, allowing assessment of genotype relations to quantitative AD pathology and cognitive function proximate to death. We subsequently leveraged the detailed phenotypes available in our cohorts to dissect the functional pathways that link genetic variants to cognitive impairment.

Ethics Statement
Written informed consent and an Anatomic Gift Act were signed by all Religious Orders Study and Rush Memory and Aging project participants after the procedures were fully explained, and both studies were approved by the Institutional Review Board of Rush University Medical Center. The work described in this report was additionally approved by the Institutional Review Boards of the Brigham and Women's Hospital and Massachusetts Institute of Technology.

Subjects
Clinical and post-mortem data came from participants in the Religious Orders Study and Rush Memory and Aging Project, two longitudinal, epidemiologic clinical-pathologic studies of aging and AD [17]. In both studies, participants without known dementia at baseline agreed to annual detailed clinical evaluation and brain donation at the time of death. Participants in the Religious Orders Study were older Cathololic nuns, priests and brothers from about 40 groups in 12 states across the United States. Subjects in the Rush Memory and Aging Project were older, community-dwelling persons from about 40 retirement communities and subsidized senior housing facilities across northeastern Illinois. Since 1993, more than 2,300 persons agreed to participate in these studies. The overall follow-up rate exceeds 90% of survivors and the overall autopsy rate exceeds 90% of decedents. Of those subjects with completed neuropathologic analyses, and following genotyping quality control filters, 414 persons with genotyping data were available for analysis in February of 2009 when this study was initiated (250 from the Religious Orders Study and 164 from the Rush Memory and Aging Project).

Clinical evaluation
The clinical diagnoses of dementia and AD were made each year following the recommendations of the joint working group of the National Institute of Neurologic and Communicative Disorders and Stroke and the AD and Related Disorders Association [27], as previously described in detail [28]. Probable AD refers to persons with clinical AD and no other clinical condition contributing to cognitive impairment and possible AD refers to persons meeting inclusion criteria for AD who are thought to have another condition (e.g., stroke) contributing to cognitive impairment. MCI referred to those individuals rated as cognitively impaired by the neuropsychologist but not demented by the examining physician, as previously described [29]. At the time of death, clinical data were reviewed by a neurologist without access to post-mortem data and a summary diagnostic opinion was rendered regarding the most likely clinical diagnosis at the time of death. Level of cognition was based on cognitive testing performed proximate to death. The Religious Orders Study and Rush Memory and Aging Project have 19 cognitive performance tests in common, and use identical analytic procedures to develop summary statistics. Mini-Mental State Examination [30] was used to describe the cohort and one test was used for diagnostic classification purposes only. The remaining 17 tests have been previously described [17]. Tests were converted to z scores, using the mean and SD from the baseline evaluation of all participants, and averaged to yield summary measures of global cognition and five cognitive domains: episodic memory, semantic memory, working memory, perceptual speed, and visuospatial ability. Summary measures minimize floor and ceiling effects and other sources of random variability. For the mediation analyses incorporating diagnosis of diabetes, annual clinical evaluations allowed documentation of history of diabetes and use of medications to treat diabetes. Diabetes was determined to be present if the participant was ever taking a medication, such as insulin or an oral hypoglycemic, to treat diabetes, as determined by direct inspection of medication containers, or ever reported a history of diagnosis of diabetes, or both, as previously described [31].

Neuropathological evaluation
Brain autopsies were performed across the US as previously described [17]. Bielschowsky silver stain was used to visualize neuritic plaques, diffuse plaques, and neurofibrillary tangles in tissue sections from the midfrontal, middle temporal, inferior parietal, and entorhinal cortices, and the hippocampal CA1 sector. The neuropathologic diagnosis of AD was made by a board-certified neuropathologist without access to any clinical data as previously reported [17,28]. We classified persons as having pathologic AD based on intermediate or high likelihood of AD by National Institute on Aging (NIA)-Reagan criteria using CERAD estimates of neuritic plaque density and Braak staging of neurofibrillary pathology [32][33][34], as previously described [17]. The quantitative composite AD pathology score was based on counts of neuritic plaques, diffuse plaques and neurofibrillary tangles as previously described [35,36]. Because the means, standard deviations, and ranges of the data varied widely for the pathologic indices, we converted the raw counts to a standard distribution by dividing each person's count by the standard deviation for that particular count and formed a summary measure by averaging the scaled scores. Because the data were skewed, square root of the scaled score was used in analyses. Separate summary measures of neurofibrillary tangles and neuritic and diffuse plaques were also made. Chronic macroscopic cerebral infarctions and alpha-synuclein immunoreactive Lewy bodies were determined as previously described and considered present or absent for analyses [17].
Genotyping DNA was extracted from lymphocytes or frozen post-mortem brain tissue. APOE genotyping was performed by Agencourt Bioscience Corporation (Beverly, MA) utilizing high throughput sequencing of codon 112 (position 3937) and codon 158 (position 4075) of exon 4 of the APOE gene on chromosome 19. In addition to the APOE e4 and e2 alleles, 32 SNPs were selected for genotyping in our cohort, based on prior evidence from the literature, as of February, 2009. Thus, the more recently discovered CR1, CLU, and PICALM [15,16] loci were not included in this study, but are the focus of a separate study (Chibnik et al., submitted). The selected SNPs were equally divided between the top results of AD case/control GWA studies [5][6][7]9,[10][11][12][13][14] (16 SNPs) and candidate gene association studies (16 SNPs), which were chosen based on their top ranking in AlzGene meta-analyses [2]. The 32 candidate SNPs were genotyped using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry on a MassARRAY platform (Sequenom). After excluding subjects for failed genotyping exceeding the 10% threshold, 414 individuals remained for subsequent analysis (genotyping rate in these subjects was .99%). All SNP allele frequencies satisfied Hardy-Weinberg equilibrium (p.0.001). Allele frequencies were not significantly different between the Religious Orders Study and Rush Memory and Aging Project subjects, supporting the validity of pooled analyses.

Statistical Analysis
Given the complementary study designs and similar procedures for data collection and generation of the cognitive and neuropathologic outcome traits, we pooled data from the Religious Orders Study and Rush Memory and Aging Project for our analyses, consistent with numerous prior studies [17,23]. Genetic association was performed using the PLINK analysis software toolkit [37]. Linear regression was used to evaluate the association of allele genotypes with level of cognition proximate to death in a 2-degree-of-freedom, genotypic test of association, with covariates included for age, gender, and years of education. In order to refine the genetic model, we additionally tested selected SNPs using a 1degree-of-freedom test to examine for additive, dominant, or recessive allelic effects. These studies were performed using PLINK as well as the R statistical computing program (www.rproject.org). Linear regression modeling in R was used to calculate residual quantitative trait variance explained, and to perform statistical mediation analyses. For the case/control association analysis based on AD clinical diagnosis, logistic regression was performed in PLINK under both additive and genotypic models, and again including covariates for age, gender, and education. All p-values reported are unadjusted for multiple hypothesis testing. A Bonferroni-corrected significance threshold of p,0.001 was calculated for the 34 SNPs tested for associations with our two primary outcomes, a quantitative measure of global cognitive performance and global AD pathology. Given the high correlation between the pathologic and cognitive traits, applying an adjustment for 68 tests would be overly conservative. Otherwise, the threshold of p,0.01 was selected to indicate suggestive statistical evidence of association. All other evaluated phenotypes, including those for AD clinical diagnosis, pathology sub-types, and cognitive domains were considered secondary analyses.

Associations with global AD pathology and global cognition
Subject demographics, clinical and neuropathologic diagnoses, cognitive status, and APOE genotypes for the cohort analyzed in this study are presented in Table 1. In clinical evaluations proximate to death, of the 414 subjects in our study cohort, 131 (31.6%) had normal cognition, 98 (23.7%) had mild cognitive impairment, and 185 (44.7%) were demented (173 met criteria for possible or probable AD). As expected, a significant proportion (41.5%) of individuals without dementia satisfied NIA-Reagan pathological criteria for intermediate or high likelihood AD.
We initially tested for associations between each of the 34 polymorphisms and our two primary outcomes, intermediate phenotypes representing a measure of global AD pathologic burden on autopsy and a measure of global cognitive function proximate to death ( Table 2). Linear regression models were used to examine the relation of SNP genotypes to the quantitative neuropathologic and cognitive traits in a 2 degree-of-freedom statistical test, adjusting for the effects of age at death, gender, and years of education. As expected, APOE e4 was significantly associated with both cognition (p = 3.4610 210 ) and AD pathology (p = 1.6610 224 ) in our cohort, whereas an association with APOE e2 was only seen for the pathological phenotype (p = 9.1610 24 ). In addition, we found associations with AD intermediate phenotypes for two SNPs, within the zinc finger protein 224 (ZNF224) and phosphoenolpyruvate carboxykinase 1 (PCK1) genes, both of which were selected for genotyping based on their identification in AD case/control GWA studies [6,10]. The ZNF224 SNP (rs3746319) was associated with both global cognition (p = 0.009) and global AD pathology (p = 0.004). In contrast, the PCK1 SNP (rs8192708) was significantly associated with global cognition (p = 3.57610 24 ) but not global AD pathology (p = 0.056), suggesting that this locus may influence cognitive impairment through mechanisms other than AD pathology. Besides APOE e4, none of the SNP associations surpass the currently accepted threshold for genome-wide significance (p,5.0610 28 ); however, the association between PCK1 and global cognition exceeds a Bonferronicorrected significance threshold of p,0.001 for 34 independent tests. Given the high correlation between the pathologic and cognitive traits, applying an adjustment for 68 tests would be overly conservative; however, the PCK1 association still exceeds that standard (p,7610 24 ). Although the risk alleles for the associations of both the ZNF224 and PCK1 loci with the intermediate phenotypes in our cohort also increase risk of AD diagnosis (Table 3), their effects are opposite to that reported in the original GWA studies [6,10]. In the case of ZNF224, we find that the minor allele, rs3746319 A , is associated with both increased AD pathologic burden and decreased cognitive performance; whereas this variant was protective against AD in the GWA study (G. Beecham and M. Pericak-Vance, personal communication). Similarly, for PCK1, the minor allele, rs8192708 G , significantly protected against cognitive decline in our cohort but was in fact the AD risk allele in the original GWA study [2]. Interestingly, two subsequent replication analyses of rs8192708 documented associations of decreased AD risk with the minor allele, consistent with our findings [4,38]. Therefore, while the effects of the ZNF224 and PCK1 loci on a diagnosis of AD and on intermediate phenotypes are consistent within our study (and in two other PCK1 replication studies); they are not consistent with the original GWA analyses. In the discussion section, we further address possible explanations for these discrepancies.
Associations with neuritic and diffuse plaques, neurofibrillary tangles, and cognitive subdomains The global AD pathology score averages the post-mortem density of neuritic and diffuse plaques and neurofibrillary tangles in multiple brain regions; however, we hypothesized that certain AD susceptibility loci might selectively promote one type of pathology, in which case the composite pathologic outcome might dilute statistical power to detect associations. We therefore performed secondary analyses to determine whether any of the candidate SNPs tested demonstrate selective or more robust association signals with separate quantitative measures of plaques or tangle pathology (Table 4). All analyses were again performed using linear regression models to test for associations with SNP genotypes, adjusted for the effects of age, gender, and education. A SNP at the GALP locus (rs3745833) showed suggestive evidence for association with diffuse plaques (p = 0.003), but not with neurofibrillary tangles (p = 0.373). In contrast, the ZNF224 SNP (rs3746319) was strongly associated with neurofibrillary tangle burden (p = 1.49610 24 ), whereas no significant association was seen with either neuritic plaque (p = 0.018) or diffuse plaque (p = 0.290) pathology. Therefore, the association with the tangle subscore is likely the primary driver for the ZNF224 locus association with global AD pathology (p = 0.009), and the composite score appears to dilute statistical power. Interestingly, the PCK1 SNP (rs8192708), which was not associated with the global pathology measure, did show suggestive evidence for association with neuritic plaque pathology (p = 0.007); however, this did not appear to explain the strong association with global cognition (p = 3.57610 24 ), as investigated further below. Similar to the approach taken with the pathological phenotypes, we performed secondary analyses to assess whether any of the SNPs showed more robust association with the five cognitive subdomains that comprise the global cognition score. Linear regression was again used to test for association of each SNP with separate quantitative trait outcomes representing episodic memory, semantic memory, working memory, perceptual speed, and visuospatial ability (Table 5). Episodic memory impairment, the most characteristic cognitive deficit of AD, was associated with both the ZNF224 locus (p = 0.003) and the PCK1 locus (p = 3.69610 24 ). ZNF224 was additionally associated with decline in visuospatial function (p = 0.007), and PCK1 showed evidence for association with semantic memory impairment (p = 0.001).

Divergent pathways from genes to cognitive impairment
For the SNPs at the ZNF224 and PCK1 loci, we performed additional linear regression analyses to refine the genetic model for the relation with our intermediate phenotypes (additive, dominant, or recessive), better characterize the strength of the observed effects, and develop statistical models to test hypotheses about mechanistic pathways. Our core regression model, consisting of age at death, gender, and years of education, explained 3% and 7% of the variation in our pathological and cognitive traits, respectively. Using the optimal dominant model of inheritance, the ZNF224 SNP (rs3746319) explained an additional 2% (Beta = 0.13, p = 0.003) of the residual variance in global AD pathology and 2.1% (Beta = 20.39, p = 0.002) of the variance in global cognition (Table 6). We next explored whether the effect of this locus on AD pathology might mediate its association with cognition (Table 7). When a term for global AD pathology was incorporated in our linear regression model, the magnitude of the association between ZNF224 and global cognition was attenuated by 44% (Beta = 20.22, p = 0.05). In our analyses of the neuropathologic subtypes, we found that the association of ZNF224 on AD pathology appeared to be due to a predominant effect on neurofibrillary tangles. Indeed, when we substituted a term for neurofibrillary tangles instead of the global pathology variable in our regression model, the effect of the ZNF224 variant on global cognition was reduced by 64%, and was no longer significant (Beta = 20.14, p = 0.21), whereas tangles showed a robust association with cognitive impairment (p,2610 216 ). These results are consistent with a sequence of events whereby an effect on the formation of neurofibrillary tangles accounts for the association of the ZNF224 allele with cognitive function. In contrast to ZNF224, the PCK1 locus showed a relatively selective association with global cognition, but not with global pathology. For this locus, an additive model of inheritance was a best fit for our data, and PCK1 explained 3.4% (Beta = 0.49, p = 1.02610 24 ) of the variance in global cognition proximate to death. We next used multiple linear regression to test whether the association of this SNP on cognitive impairment is predominantly independent of AD pathology. Indeed, after inclusion of a model term for global AD pathology, the PCK1 SNP (rs8192708) remained associated with global cognition (Beta = 0.37, p = 0.002), despite the strong, independent association between pathology and cognition (Beta = 21.22, p,2610 216 ; Table 7). Given our finding of an association with neuritic plaques, we substituted a model term for neuritic pathology instead of the global pathology variable; however, despite a modest reduction in the effect size, the relation between PCK1 and cognitive impairment remained significant (Beta = 0.31, p = 0.005; Table 7). Besides AD-related pathology, Lewy bodies and infarcts are the two additional brain pathologies most commonly seen in association with age-related cognitive decline [39]. We therefore investigated whether the association of PCK1 on global cognition might be mediated by either Lewy bodies or infarcts, by including relevant terms into our regression model (Table 8). Again, the association between PCK1 and global cognition remained  , and PCK1 continued to explain 3% of the residual variance in global cognition in our cohort after adjusting for the three most common brain pathologies associated with dementia. Common variation at the PCK1 locus has also been associated with type 2 diabetes in a number of independent studies [40][41][42]. Since diabetes has also been implicated as a risk factor for age-related cognitive decline [31], we examined whether diabetes mediates the association of PCK1 with cognitive impairment. However, adjusting for diabetes diagnosis in our linear regression model did not significantly attenuate the association of PCK1 with global cognition (Beta = 0.370, p = 0.001).

Discussion
In this study, by genotyping a panel of loci within two cohorts of subjects with detailed cognitive and neuropathological characterization, we evaluate intermediate phenotypes as a tool for the functional dissection of candidate AD susceptibility loci. Using the extensively validated APOE locus, we previously demonstrated that intermediate traits enhance statistical power to detect associations, even in studies of modest sample size. Here, using the same strategy, we present evidence supporting the possible role of two additional loci in influencing age-related cognitive decline and AD neuropathology. Specifically, the ZNF224 locus is associated with a quantitative measure of global AD pathology, and both ZNF224 and PCK1 are associated with a summary measure of global cognition proximate to death. Using separate quantitative traits for each of the predominant AD pathological features, we document associations between GALP and PCK1 and diffuse and neuritic plaque pathology, respectively, whereas ZNF224 showed a relatively selective association with neurofibrillary tangle pathology. Finally, in a series of statistical mediation analyses, we tested hypotheses about the causal chain of events linking genetic variation in the ZNF224 and PCK1 loci with cognitive decline, with strikingly different outcomes. In the case of ZNF224, we find that AD pathology, and more specifically, neurofibrillary tangles mediate an association with cognitive impairment. In contrast, we find that the association between PCK1 and cognition is largely independent of not only AD pathology, but also Lewy bodies, and infarcts, which together comprise the three most common known brain pathologies associated with dementia [39,43].
Both ZNF224 and PCK1 were initially implicated by AD GWA studies; however, neither locus has yet been consistently replicated in subsequent genetic studies, and little is known about their potential mechanism of action in disease pathogenesis. The ZNF224 locus encodes a Kruppel-associated box-containing zinc-finger protein that is widely expressed, including in the adult brain, and likely functions as a transcriptional repressor [44,45].
The SNP evaluated in this study, rs3746319, encodes a missense mutation causing a Lys to Glu change at position 640, which falls near the C-terminus within one of 19 zinc-finger repeat motifs. However, we do not yet know enough about ZNF224 protein structure and function to speculate further on how this variant might promote neurofibrillary tangle formation and subsequent cognitive impairment, and further investigation will be required to determine if rs3746319 is the causal variant and whether ZNF224 is indeed the causal gene. The PCK1 gene encodes phosphoenolpyruvate carboxykinase 1, which catalyzes the rate-limiting step of gluconeogenesis [46]. The SNP genotyped in our study, rs8192708, is also a missense mutation, causing an Ile to Val change at position 267; however, the functional consequences of this change, if any, are not known. PCK1 variants have also been suggested to be associated with diabetes [40][41][42], and independently, diabetes has been identified as a risk factor for the development of dementia [31]. In our mediation analysis, adjusting for the effect of diabetes diagnosis did not account for the association of PCK1 and cognitive impairment; however, it is possible that an appropriate intermediate phenotype, such as direct measurements of blood glucose or hemoglobin A1c, might allow detection of mediation. In another study performed in the same cohort, a relation was found between diabetes and infarcts [47]; however, we were also unable to mediate the PCK1 association by including a model term for cerebral infarctions. Our finding that the PCK1 association with cognitive decline is not explained by AD pathology, Lewy bodies, or infarcts suggests that this locus might influence additional, unmeasured pathologies. For example, whereas our analyses adjusted for macroscopic infarcts, PCK1 may instead primarily influence microscopic forms of cerebrovascular injury. Further, while our intermediate pathologic phenotype accounts for amyloid plaques and neurofibrillary tangles, it does not capture levels of soluble, but potentially still neurotoxic, forms of amyloid or tau pathology [48,49]. Alternatively, variation at PCK1 might influence one or multiple steps in the cascade of events predicted to occur downstream of amyloid, tangles and other pathologies, such as synapse loss, inflammation, and/or cell death pathways.
Unexpectedly, the variants in ZNF224 and PCK1 show opposite directions of allelic effects for association with AD intermediate phenotypes in our cohort compared to their association with AD diagnosis in the initial GWA studies. In other words, the alleles associated with increased AD risk in the initial reports (rs3746319 G and rs8192708 G ) are actually protective against cognitive decline in our cohort. Importantly, this discrepancy is not accounted for by our use of intermediate phenotypes, as the ZNF224 and PCK1 SNPs show consistent direction of affect on AD diagnosis in our study population (Table 3). Such ''flip-flop'' associations have been reported with increasing frequency as GWA scans are completed for many common diseases, and replication efforts are subsequently undertaken [50]. Indeed, in the case of PCK1, two prior replication studies found evidence that the major allele, rs8192708 A , may increase risk for dementia, consistent with our results suggesting an association between this allele and both cognitive decline and AD [4,38].
The interpretation of reversals in the direction of variant associations between different study cohorts remains controversial [50]. The most common explanation for such observations are that they are in fact spurious and representative of chance fluctuations around the null hypothesis. However, in our study, the strongly suggestive statistical evidence for the associations between ZNF224 and PCK1 with AD intermediate phenotypes makes their arising by chance less likely; and additionally, the reversals of allelic effect are seen with both loci in our analysis. Instead, we propose that differences in subject ascertainment and recruitment are more likely to be responsible for our observations. The Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP), from which our study cohort is based, are prospective, longitudinal studies in which subjects from the community are recruited non-demented at baseline (mean age = 75 and 79, for ROS and MAP respectively). All cases of clinical AD are therefore incident within our cohort. In contrast, similar to nearly all AD GWA studies performed to date, the initial reports of association with the PCK1 and ZNF224 loci come from AD cases recruited from a neurology clinic population with prevalent dementia. In addition, whereas subjects in our study were recruited at approximately similar ages to the GWA cohorts, they were significantly older at the time of last clinical evaluation and autopsy (mean age of death = 87). Studies with different designs (cross-sectional vs. prospective) and varying methods of subject ascertainment can generate contradictory epidemiological findings, for example due to survival bias. If an AD risk allele is associated with earlier age of dementia onset; it might be underrepresented in the prospective cohort, which requires subjects to be non-demented at enrollment; and therefore, might subsequently appear to be a protective allele. ''Flip-flop'' associations might additionally arise from variation in linkage disequilibrium structure in the genomic region of interest between the cohorts in different studies. In fact, both the ZNF224 and PCK1 SNPs fall under modest recombination peaks, based on HapMap data [51]. Although both our study and the GWA analyses were conducted in subjects of European ancestry, it remains possible that sampling variation between two populations of similar ethnicity might lead to the association reversal that we have observed, as recombination could distribute our tag SNP onto haplotypes that are different from that harboring the causal variant [50]. Ultimately, further analysis of both SNPs and fine mapping of each locus in larger study samples will be required to validate both PCK1and ZNF224 as AD susceptibility loci, and resolve which allele may increase risk for disease. Of the thirty-four SNPs evaluated in our study, both of the loci that we found to be associated with AD intermediate phenotypes were initially identified by GWA studies, suggesting the power of this unbiased approach to identify genes that might be overlooked by prevailing hypotheses of disease biology. Our study was initiated prior to the recent report of two large AD case/control GWA studies which independently identified three new susceptibility genes, CLU, CR1, and PICALM [15,16]. In a parallel effort, we recently found that CR1 is associated with age-related cognitive decline in our study cohorts; and further, that this association was mediated by an effect on amyloid pathology (Chibnik et al., submitted). The power of a GWA study design and the types of genes one expects to discover are tightly linked to the selected phenotypic outcome. To the extent possible, the chosen outcome measure should be closely matched to the underlying biology responsible for the heritable trait variation of interest. In autopsy cohort studies of aged individuals in the community setting, most subjects with probable AD demonstrate multiple brain pathologies [52]. Based on our results, we believe that intermediate pathological and cognitive traits have great promise to enhance gene discovery and for functional characterization of loci that emerge from current AD GWA studies.


extfieldsofstudy: Biology Medicine

text:
The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

Background The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. Results A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. Conclusions The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.

Background
HIV-1, the retrovirus responsible for the current worldwide AIDS pandemic, is an enveloped virus. The envelope protein (Env) of HIV-1 is essential for determining host range and for inducing the membrane fusion that allows the virus to enter the host cell. The former and latter functions are mediated by the SU (gp120) and the TM (gp41) subunits of the envelope protein, respectively [1][2][3]. The SU and TM are generated from a precursor (gp160) by cellular proteases that recognize a basic amino acid sequence between gp120 and gp41 [4][5][6].
This proteolytic processing is essential to generate fusion-competent HIV-1 Env and is believed to take place in an early Golgi region [7,8].
HIV-1 Env is anchored across lipid bilayers via its highly conserved membrane-spanning domain (MSD) [9]. Although the possibility of a transient alteration of the membrane topology exists [10,11], HIV-1 Env is widely believed to be a type I membrane protein with a single α-helical MSD in the steady state [12]. Two different models exist within the single MSD model of HIV-1 Env. In an initial model, the MSD is supposed to be 23 amino acid residues long, ranging from Lys683 to Val704 in the HXB2 sequence, and has a highly conserved hydrophilic arginine residue in the midst of its hydrophobic amino acid sequence [13]. In an alternative model, MSD is shorter; and the arginine residue in the lipid bilayer is expected to interact with the polar head of the lipid molecule [14,15].
The primary structure of the MSD of HIV-1 Env also has a GXXXG motif, a motif often found at the helixhelix interface of transmembrane α-helices [16]; it exists upstream of the arginine residue. If an ordinary α-helix structure is assumed for the MSD, the GXXXG motif and arginine residue are positioned on opposite sides of the gp41 MSD α-helix.
In vitro studies of the gp41 MSD showed a high tolerance for mutations. For example, the above mentioned conserved arginine residue [17] and the GXXXG motif can accommodate point mutations [18]. Even several heterologous MSDs can replace the entire gp41 MSD without deteriorating effects [17,19]. These findings led to the notion that the specific amino acid sequence in the gp41 MSD has no significant biological role within the limits of the assays used. This is a curious notion since the sequence is quite conserved in nature, despite the virus being subject to very strong sequence diversification from errors in reverse transcription.
In fact, other studies have suggested that the specific sequence of the gp41 MSD plays a role in the function of gp41 [20,21]. We have shown that replacing the gp41 MSD with MSDs derived from glycophorin A or VSV-G, each containing the GXXXG motif, severely decreases the fusion activity of HIV-1 Env [18,22]. Furthermore, simultaneous substitution of all three glycine residues, within the GXXXG motif with leucine residues, also negatively affected the function of the HIV-1 Env [23]. Shang et al. recently showed the importance of the GXXXG region using a unique genetic approach [24]. These studies clearly suggested the presence of important information encoded in the sequence of MSD. However, the nature of the code is still not evident.
To further elucidate the structure-function relationship of the gp41 MSD, we analyzed a circular dichroism (CD) profile of the synthetic peptide corresponding to the MSD and obtained the profile expected for α-helical secondary structure. Next, we used the envelope gene of HXB2 [25] to create a series of alanine insertion mutants of the entire predicted MSD. We found that alteration of the relationship between Gly694 and Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis of the MSD α-helix resulted in fusion incompetent Env. These mutant Envs also showed defects in proteolytic processing and intracellular transport in the endoplasmic reticulum (ER) and Golgi regions. We further showed that the intracellular transport of HIV-1 Env is regulated by the MSD region, through experiments that transplanted the gp41 MSD into another membrane protein, Tac.
This transplantation led to an alteration of the intracellular distribution of Tac, similar to that of HIV-1 Env.

Results
Circular dichroism analysis of the synthetic MSD peptide in lipid shows a-helical secondary structure The primary structure of the gp41 MSD is highly conserved, and its secondary structure has been predicted to be an α-helix based on computational algorithms [26]. However, there are no physical data to support this expectation. We synthesized a peptide corresponding to a consensus HIV-1 clade B structure of the gp41 MSD and determined its CD spectrum in lipid bilayers. The CD profile, shown in Figure 1, has negative maxima near 208 nm and 222 nm, indicating the presence of an α-helical structure. Although the gp41 MSD of HIV-1 contains three glycine residues, thought to be helixbreaking residues in soluble proteins, the dominant structure indicated by our CD data was an α-helix. Many glycines are found in transmembrane helices. Addition of lysine residues at both ends was necessary to allow us to purify the extremely hydrophobic MSD peptide. We cannot completely exclude the possibility that these lysine residues at the termini, especially at the C-terminus, may stabilize the α-helical structure.
Scanning alanine-insertion mutagenesis identified the region of gp41 MSD critical for membrane fusion To identify the region of the gp41 MSD α-helix critical for its function, we generated a set of alanine-insertion mutants covering the entire predicted MSD by using the HXB2 envelope gene. The alanine residue was chosen because it can be well accommodated in an α-helix [27,28]. Since previous data suggest the involvement of the gp41 MSD in membrane fusion [18,23,24,29], membrane fusion activity was determined for the mutants. The primary structures of these mutants are shown in Figure 2. Nomenclature is based on the positions of the inserted alanine residues in HIV-1 Env. Therefore, 684 +A mutant indicates that the inserted alanine residue corresponds to the 684th residue of the envelope protein. The mutant envelope gene was cloned into the envelope expression vector, and the fusion activity of each mutant was determined by the T7 RNA polymerase transfer assay as described previously [18]. The result is shown in Figure 3A. Among the twenty-two mutants we generated, three showed a prominent decrease in the fusion activity. These three are 694+A, 695+A, and 696+A; their relative fusion activities when compared with the wild type (WT) were 37.5%, 14.0% and 15.5%, respectively. Mutants 695+A and 696+A showed more severe defects than 694+A. Thus the corresponding region from 694 to 696, the G 694 LR 696 region, was shown to be critical for fusion activity.
The alteration of the phase of the GLR region in MSD was critical to membrane fusion The insertion of an alanine residue affects both the length and the phase of the α-helix. However, we expected that the local phase might have a more important role than the length of the MSD because all the insertion mutants generated are expected to have the same length of the MSD. To verify this, we inserted one, two, three, and four alanine residues between residues 694 and 695 (Figure 2 bottom, 695+2A, 3A, and 4A) and examined the fusion activities of each resulting mutant. The result is shown in Figure 3B. The insertion of two residues caused a further decrease in the fusion activity compared to the single insertion (compare 695 +A and 695+2A). However, the fusion activity slightly recovered with the insertion of three residues, and almost fully recovered by the insertion of four alanine residues. It seems that there is a correlation between the recovery of the phase of gp41 MSD α-helix and recovery of membrane fusion activity. The observed defect in fusion activity was not due to the increase in length but instead to the local shift of the gp41 MSD α-helix.
To further identify residues critical for determining the phase in the GLR region, we generated 696+2A and 695/696+2A (a combination of 695+2A and 696+2A, Figure 2 bottom) and then compared the fusion activity together with 695+2A. Both 695+2A and 696+2A showed severe defects in membrane fusion ( Figure 3C). Interestingly, the combination of these two (695/696 +2A) recovered fusion activity. The phase commonly altered in the fusion defective mutants, 695+2A and 696 +2A, but corrected in the fusion competent 695/696+2A mutants was found to be between Gly694 and Arg696. Thus the relationship between Gly694 and Arg696 seems to be an important factor for the membrane fusion activity.
Analysis of the protein profile of the fusion-defective mutants reveals impaired processing of gp160 into gp120 and gp41 We analyzed the protein profiles of these mutant Envs by immunoblotting, using anti-gp120 and anti-gp41 antibodies ( Figure 4). All mutant Envs were expressed at comparable levels ( Figure 4A); however, the fusiondefective mutants had impaired processing of gp160, namely more gp160 than gp120; and accordingly less gp41 (see 694+A, 695+A, and 696+A) was observed. This tendency was more prominent for 695+2A and 696 +2A, each of which showed severe defects in fusion. A similar correlation between impaired processing of Env and defective membrane fusion was observed in the multiple alanine insertion mutants that showed defective fusion ( Figure 3C and 4B). Because the generation of processed gp41 is a prerequisite for fusion competency, this protein profile for inefficient gp160 processing is consistent with the observed fusion defect. Our data showed that the alteration in the α-helical phase in the localized region within gp41 MSD affected processing of gp160 into gp120 and gp41. It was also shown that these mutants were fusion incompetent. A possibility is that the mutations induced allosteric structural changes of the cleavage site so that the mutant Env was no longer processed properly by Furin or Furin-like proteases. However, this idea was not supported by the observation that mutant gp160, purified from COS-7 cells, is cleaved into the gp120 and gp41 subunits by commercially available Furin in vitro (Additional file 1). We also analyzed the trimerization of Env mutants.
The trimer of 695+2A Env was detected (Additional file 2) However, the presence of less drastic yet critical structural alterations by the mutation cannot be ruled out completely.
Alanine insertion in the gp41 MSD can alter the intracellular distribution of Env Since processing of gp160 takes place in the Golgi [7,8], we hypothesized that the defect in the processing was derived from the defect in the intracellular trafficking of mutant Env in the endoplasmic reticulum and Golgi regions. To test this possibility, we examined the distribution of mutant Env in the cells. We attached a FLAG tag at the C-terminus of gp41, providing a linear epitope that can be recognized by monoclonal antibody, M2. An ttachment of the FLAG tag did not alter the defect in processing present in alanine insertion mutants (data not shown). The envelope proteins expressed in COS-7 cells were visualized by immunofluorecent assay using the anti-FLAG monoclonal antibody. We observed that fine, mesh-like fluorescent signals distributing within the transfected cells were more prominent for the mutant 695+2A than the WT ( Figure 5). The intensity of fluorescence derived from Env at the Golgi area was notably weaker for 695+2A than for the WT. These data suggested that mutant Env was defective for transport from ER to Golgi. The level of Env expressed on the cell surface, analyzed by FACS, is consistent with this observation because it is lower for the mutant than for the WT ( Figure 6).
To further verify the transport defect biochemically, we analyzed the pattern of modification of sugar moieties in the WT and mutant Env. The results are shown in Figure 7. When treated with endoglycosidase H (Endo H), the WT exhibited an Endo H-resistant fraction of gp160 whereas almost no Endo H-resistant gp160 was detected in the 695+2A mutant. This finding indicated that sugar moieties attached to the mutant envelope protein remained as high-mannose types. However, both the WT and mutant envelope proteins generated bands that migrated similarly after treatment with Peptide: N-Glycosidase F (PNGase F), which cleaves between the innermost GlcNAc, and asparagine residues, where sugar moieties are attached. These data further confirmed the defect of the mutant envelope protein in transport, probably in ER-Golgi regions.
The transfer of the gp41 MSD into a foreign membrane protein alters the intracellular distribution of chimeric proteins We are interested in determining whether the MSD region alone is sufficient to induce the observed transport defect in the context of other membrane proteins. To test this possibility we have replaced the MSD of Tac, the α-chain of the Interleukin-2 receptor, with the MSD of the wild type (Tac-gp41WT) or 695+2A mutant (Tac-gp41+2A) of gp41, and determined the intracellular distribution of the engineered Tac proteins. We included the intact Tac as a reference (Tac-WT). The results are shown in Figure 8. The signals of intact Tac proteins distributed both in the cytoplasm and plasma membrane areas. They show a fine mesh like appearance in the cytoplasm and are well overlapped with the signals of the ER markers. The intact Tac proteins also showed prominent signals at the rim of the cells suggesting efficient transport to the plasma membrane ( Figure 8A and 8D). There was no overlap of signals for intact Tac and Golgi markers ( Figure 8G). When the MSD of intact Tac proteins was replaced with that of gp41 (wild type in Figure 8B and 8E; 695+2A in C and F), the signals corresponding to the plasma membrane areas became weaker than those of intact Tac (Figure 8, compare A with B and C; D versus E and F). The majority of the signals was observed in the cytoplasm, and the signals were co-localized with ER markers (Figure 8B and 8C). There are some signals of Tac-gp41 chimera in Golgi areas ( Figure 8H and 8I). Different from the context of HIV-1 envelope proteins ( Figure 5E and 5F), we did not detect a discernable difference in the distribution between the wild type gp41 MSD (Figure 8 Tac-gp41WT) and 695+2A gp41 MSD (Tac-gp41+2A) in the Golgi areas ( Figure 8H and 8I). It appeared that the introduction of the gp41 MSD made chimeric Tac distribute in the cytoplasmic region, mainly ER regions, but the difference between the wild type gp41 and 695+2A mutant became less prominent in the context of Tac than in the context of the HIV-1 Env.

Discussion
Although the gp41 MSD has three glycine residues, our CD analysis suggested the presence of the α-helical structure in gp41 MSD (Figure 1). This may not be a surprise, since glycines are abundant in transmembrane helices and glycines are viewed as helix breakers in soluble proteins. A recent molecular dynamics study also supports a helical conformation [30]. Furthermore, the replacement of all three glycine residues with alanine residues, highly α-helix-forming residues [27,28], did not affect the fusion activity of gp41 [18]. Thus gp41 MSD is presumably functional with an α-helical structure. These data, however, do not rule out the possibility of the reported transient alteration of the secondary structure of the gp41 MSD during membrane fusion [11].
Our scanning alanine insertion mutagenesis identified the topological relationship between Gly694 and Arg696 around the MSD α-helix as a critical determinant for the proper processing ( Figure 4) and intracellular Figure 3 The fusion activity of Ala-insertion mutants in the cell-cell fusion assay. COS-7 cells transfected with the T7 RNA polymerase expression vector and the Env expression vector were co-cultured with 293CD4 cells transfected with a plasmid containing T7 promoter-driven renilla luciferase reporter. After a 24-hr co-culture, the renilla luciferase reporter activity was measured and normalized to the firefly activities as described previously [18]. The normalized renilla luciferase activities for (A) single Ala-inserted mutant of Env, (B) the mutant Env with multiple Ala insertion, (C) mutant Env with two alanine residues inserted at positions 695 and 696 are shown. Data are the average of three independent experiments. The error bar indicates a standard error. transport ( Figure 5) of Env. Since the processing of gp160 is dependent on the proper transport of the proteins to the Golgi apparatus, it seemed that the observed defect in processing might be due to a transport defect. However, we cannot rule out the possibility of a potential allosteric structural alteration of the Env by mutation in the MSD as a cause for the inefficient processing. Indeed our recent data suggested that the mutations in the gp41 MSD exert allosteric conformational changes of the ectodomain of HIV-1 Env [22].
Mutation at the cleavage site of gp160 eliminates HIV-1 Env fusogenicity [7]. Thus, the defective membrane fusion of our alanine insertion mutants seemed to be derived from improper processing of gp160. However, there are other factors contributing to the defective fusion. Many studies have shown that mutations in the gp41 MSD affect membrane fusion efficiency [18,23,24,29]. In the context of 695+2A mutant, the substitution of hydrophilic arginine residue with non-polar residues (alanine or isoleucine) rescues the defective processing (Additional file 3); however, this could not resolve the defective fusion (Additional file 4). These data suggest that gp41 MSD has a role(s) in the membrane fusion process itself. To reveal the exact mechanism, further studies are required.
It has been reported that MSD length is crucial for the trafficking of membrane proteins [31]. In HIV-1 Env, length of the MSD alone does not seem to be a primary determinant for trafficking. However, our data show that critical information lies in the local structure of the transmembrane α-helix of gp41. It is possible that the alteration of structural features in the MSD region can be sensed by host factor(s) involved in the protein quality control system. This detection could be through the MSD region itself. In a yeast system, some proteins involved in the vesicular transport in ER-Golgi where target recognition was achieved via the MSD region have been reported [32,33]. Since the distribution of our Tac-gp41 chimera was heavily affected by the replacement of the MSD region alone (Figure 8), it may support such a hypothesis. Such a hypothetical factor may recognize wild type gp41 MSD via the GXXXG motif facing outward in relationship to the MSD bundle, if the gp41 MSDs interact with each other through arginine residues as suggested recently [30].
Notably, our alanine insertion mutation altered the relative positioning of the GXXXG motif and arginine residue within the gp41 MSD. Both are major interaction motifs between transmembrane α-helices [34,35]. Although recent electron cryomicroscopic data [36][37][38] did not provide a spatial arrangement of the gp41 MSD portions, it is possible that there are interactions between the gp41 MSDs during the biosynthesis of the HIV-1 Env. Our alanine insertion may disrupt the interaction among MSDs. This disturbance of interhelical interactions may result in altered intracellular transport. The failure to reproduce differences in intracellular distribution between the wild type and 695+2A MSD, in the context of Tac (Figure 8, B-I), may arise from the Figure 4 The immunoblotting analysis of wild type (WT) and Ala-inserted mutant Env. The envelope proteins expressed in COS-7 cells transfected with the Env expression vector were detected with anti-gp120 antibody (for gp160 and gp120) or with anti-gp41 antibody. The results of single-and multiple-Ala-insertion mutants are shown in (A) and (B), respectively. difference in the oligomeric status between HIV-1 Env (trimer) and Tac (monomer). Our data suggest that mutant Env still forms a trimer (Additional file 2).
Our data clearly demonstrate that the MSD of gp41 has important functions in the biosynthesis of HIV-1 Env, apart from the simple anchoring and modulation of fusion efficiency. The exact regulation mechanism of intracellular distribution of HIV-1 Env by the MSD portion is not known; however, it could be of great importance to determine whether there are any cellular factors that specifically recognize the MSD region of HIV-1 Env.

Conclusions
We have shown that the secondary structure of the synthetic peptide of gp41 MSD is an α-helix. Based on this information, we performed a scanning alanine insertion mutagenesis which showed that alteration of the topological relationship between conserved GXXXG motif and the arginine residue resulted in non-functional Env. The mutant Env manifested a reduced fusion activity and impaired the processing of gp160 into gp120 and gp41. Furthermore, the intracellular transport of mutant Env was affected in the endoplasmic reticulum and Golgi areas. Our data suggested that the specific α-helical structural feature of gp41 MSD controls the biosynthesis of HIV-1 Env.

Synthesis of MSD peptides and its circular dichroism analysis
The sequence of the peptide used is KKWYIKIFI-MIVGGLVGLRIVFAVLSIVNRKK, which corresponds to the consensus sequence of predicted MSD of clade B HIV-1. The sequence of the MSD of the clade B molecular clone, HXB2, used in this study differs by one amino acid from this sequence (indicated by the underline, HXB2 has L instead of I at this position). Two lysine residues were introduced at the N-and C-termini to make the peptide more hydrophilic. The CD spectra were measured at 25°C with Aviv Model 215 (Aviv biomedical Inc, Lakewood, NJ) in 15 mM DPC (n-dodecyl pyridinium chloride), 20 mM NaPi, 150 mM NaCl. The concentration of the peptide was 10 μM. Eight spectra were averaged after subtracting for a DPC reference sample.

Generation of the MSD mutants
QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) generated the mutants used in this study. The plasmid, pGEM7zNB, which contains the 1.2-kb NheI-BamHI fragment covering the env portion of HXB2RU3ΔN, was used as a template as described previously [18]. To facilitate the mutagenesis, silent restriction The NheI-BamHI fragment of pGEM7zNB containing the expected mutations was cloned back to pElucEnv [18] or pElucEnv-3FLAG Env (see below) expression vectors.  The synthetic codon-optimized gene corresponding to the Tac protein, α-chain of Interleukin-2 receptor, with the gp41 MSD was custom synthesized (GenScript, Piscataway, NJ). The derivatives of this construct, whose MSD portion was replaced with those of wild type or mutant gp41 or intact Tac, were generated by mutagenesis using PCR. These genes were cloned downstream of the CMV promoter to generate the Tac-derivative expression vectors.
Addition of the 3 × FLAG tag at the C-terminus of the Env A 3 × FLAG tag was added to the C-terminus of gp41 by inserting oligonucleotides corresponding to the 3 × FLAG tag sequence derived from the vector p3xFLAG-CMV™-7.1 (Sigma, St. Louis, MO). Following this insertion, the amino acid sequence after the C-terminus of gp41 reads as RSARDYKDHDGDYKDHDIDYKDDDDK. The expression vector of FLAG-tagged Env was called pElucEnv-3FLAG Env. [18] were grown in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cells were kept under 5% CO 2 in a humidified incubator. Anti-gp120 polyclonal antibody was obtained from Fitzgerald Industries International, Inc. (Concord, MA). The hybridoma 902 and Chessie 8 were obtained from Bruce Chesebro and George Lewis, respectively through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA [39][40][41]. Anti-FLAG M2 and BioM2 were purchased from Sigma (St Louis, MO).

Cell-cell fusion assay
Cell-cell fusion assays, using T7 RNA polymerase (T7 RNA pol) transfer, were performed as described previously [18]. Briefly, 293-CD4 cells that constitutively express CD4 were transfected with pTM3hRL harboring the T7 promoter-driven renilla luciferase gene by FuGene 6 (Roche Applied Science, Mannheim, Germany), and were co-cultured with COS-7 cells that had been transfected with pCMMPT7iresGFP, a T7 RNA polymerase expression vector, and pElucEnv containing HIV-1 Env and firefly luciferase genes by FuGene 6. After 12 hours of co-culture, the renilla and firefly luciferase activities were measured using the Dual-Glo luciferase assay system (Promega, Madison, WI). The fusion activity, represented by renilla luciferase activity, was normalized by firefly luciferase activity to obtain transfection efficiency [18]. The polyclonal anti-halo antibody was obtained from Promega (Promega, Madison, WI).

Immunofluorescence assay
Immunofluorescence assays were used to determine the intracellular distribution of the envelope proteins. For this purpose, we generated a modified envelope expression vector called pElucEnvdeltaGFP; this is the derivative of the previously described pElucEnv [18] and it has the deletion of the EGFP portion. COS-7 cells transfected with pElucEnv WT or 695+2A in the delta GFP backbone vector and ER-DsRed2 or Golgi-YPF (Clontech) or pER-mAG1 (MBL, Nagoya, Japan) plasmid by FuGene 6 (Roche) were treated with PBS including 4% of PFA for 5 min at 48 hr posttransfection. Cells were permeabilized by PBS including, 0.05% of saponin and 0.2% of BSA, for 30 min and then stained with 20 μg/ml of bio-M2 (Sigma) antibody and 10 μg/ml of streptavidin conjugated Alexa fluor 488 or 555 (Invitrogen). In the case of Halo-tagged proteins, polyclonal anti-Halo antibodies were used as primary antibodies. The distributions of fluorescence in cells were visualized using a Zeiss LSM 510 meta confocal microscope.

Flow cytometric analysis
Flow cytometric analysis was performed as described previously [18]. Briefly, COS-7 cells were transfected with pElucEnv by FuGene 6 on a six-well plate. Fortyeight hours aftertransfection, the cells were stained with anti-gp120 monoclonal antibody 902, biotinXX antimouse IgG (Invitrogen) and streptavidin-Alexa 555 in PBS including 10% FBS. Cells were fixed with 1% paraformaldehyde in PBS and analyzed by FACS Calibur (BD Biosciences).