HIVBrainSeqDB: a database of annotated HIV envelope sequences from brain and other anatomical sites

<p>Abstract</p> <p>Background</p> <p>The population of HIV replicating within a host consists of independently evolving and interacting sub-populations that can be genetically distinct within anatomical compartments. HIV replicating within the brain causes neurocognitive disorders in up to 20-30% of infected individuals and is a viral sanctuary site for the development of drug resistance. The primary determinant of HIV neurotropism is macrophage tropism, which is primarily determined by the viral envelope (<it>env</it>) gene. However, studies of genetic aspects of HIV replicating in the brain are hindered because existing repositories of HIV sequences are not focused on neurotropic virus nor annotated with neurocognitive and neuropathological status. To address this need, we constructed the HIV Brain Sequence Database.</p> <p>Results</p> <p>The HIV Brain Sequence Database is a public database of HIV envelope sequences, directly sequenced from brain and other tissues from the same patients. Sequences are annotated with clinical data including viral load, CD4 count, antiretroviral status, neurocognitive impairment, and neuropathological diagnosis, all curated from the original publication. Tissue source is coded using an anatomical ontology, the Foundational Model of Anatomy, to capture the maximum level of detail available, while maintaining ontological relationships between tissues and their subparts. 44 tissue types are represented within the database, grouped into 4 categories: (i) brain, brainstem, and spinal cord; (ii) meninges, choroid plexus, and CSF; (iii) blood and lymphoid; and (iv) other (bone marrow, colon, lung, liver, etc). Patient coding is correlated across studies, allowing sequences from the same patient to be grouped to increase statistical power. Using Cytoscape, we visualized relationships between studies, patients and sequences, illustrating interconnections between studies and the varying depth of sequencing, patient number, and tissue representation across studies. Currently, the database contains 2517 envelope sequences from 90 patients, obtained from 22 published studies. 1272 sequences are from brain; the remaining 1245 are from blood, lymph node, spleen, bone marrow, colon, lung and other non-brain tissues. The database interface utilizes a faceted interface, allowing real-time combination of multiple search parameters to assemble a meta-dataset, which can be downloaded for further analysis.</p> <p>Conclusions</p> <p>This online resource, which is publicly available at <url>http://www.HIVBrainSeqDB.org</url>, will greatly facilitate analysis of the genetic aspects of HIV macrophage tropism, HIV compartmentalization and evolution within the brain and other tissue reservoirs, and the relationship of these findings to HIV-associated neurological disorders and other clinical consequences of HIV infection.</p

Microbial Translocation Is Associated with Increased Monocyte Activation and Dementia in AIDS Patients

Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4+ T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes

Soluble CD4 and CD4-Mimetic Compounds Inhibit HIV-1 Infection by Induction of a Short-Lived Activated State

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus–cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high–potential-energy viral entry machines that are normally activated by receptor binding

The birth of a human-specific neural gene by incomplete duplication and gene fusion

Background: Gene innovation by duplication is a fundamental evolutionary process but is difficult to study in humans due to the large size, high sequence identity, and mosaic nature of segmental duplication blocks. The human-specific gene hydrocephalus-inducing 2, HYDIN2, was generated by a 364 kbp duplication of 79 internal exons of the large ciliary gene HYDIN from chromosome 16q22.2 to chromosome 1q21.1. Because the HYDIN2 locus lacks the ancestral promoter and seven terminal exons of the progenitor gene, we sought to characterize transcription at this locus by coupling reverse transcription polymerase chain reaction and long-read sequencing. Results: 5' RACE indicates a transcription start site for HYDIN2 outside of the duplication and we observe fusion transcripts spanning both the 5' and 3' breakpoints. We observe extensive splicing diversity leading to the formation of altered open reading frames (ORFs) that appear to be under relaxed selection. We show that HYDIN2 adopted a new promoter that drives an altered pattern of expression, with highest levels in neural tissues. We estimate that the HYDIN duplication occurred ~3.2 million years ago and find that it is nearly fixed (99.9%) for diploid copy number in contemporary humans. Examination of 73 chromosome 1q21 rearrangement patients reveals that HYDIN2 is deleted or duplicated in most cases. Conclusions: Together, these data support a model of rapid gene innovation by fusion of incomplete segmental duplications, altered tissue expression, and potential subfunctionalization or neofunctionalization of HYDIN2 early in the evolution of the Homo lineage

Changes in the V3 region of gp120 contribute to unusually broad coreceptor usage of an HIV-1 isolate from a CCR5 Delta32 heterozygote

AbstractHeterozygosity for the CCR5 Δ32 allele is associated with delayed progression to AIDS in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an unusual HIV-1 isolate from the blood of an asymptomatic individual who was heterozygous for the CCR5 Δ32 allele and had reduced levels of CCR5 expression. The primary virus used CCR5, CXCR4, and an unusually broad range of alternative coreceptors to enter transfected cells. However, only CXCR4 and CCR5 were used to enter primary T cells and monocyte-derived macrophages, respectively. Full-length Env clones had an unusually long V1/V2 region and rare amino acid variants in the V3 and C4 regions. Mutagenesis studies and structural models suggested that Y308, D321, and to a lesser extent K442 and E444, contribute to the broad coreceptor usage of these Envs, whereas I317 is likely to be a compensatory change. Furthermore, database analysis suggests that covariation can occur at positions 308/317 and 308/321 in vivo. Y308 and D321 reduced dependence on the extracellular loop 2 (ECL2) region of CCR5, while these residues along with Y330, K442, and E444 enhanced dependence on the CCR5 N-terminus compared to clade B consensus residues at these positions. These results suggest that expanded coreceptor usage of HIV-1 can occur in some individuals without rapid progression to AIDS as a consequence of changes in the V3 region that reduce dependence on the ECL2 region of CCR5 by enhancing interactions with conserved structural elements in G-protein-coupled receptors

Identification and characterization of a macrophage-tropic SIV envelope glycoprotein variant in blood from early infection in SIVmac251-infected macaques

Macrophages play an important role in HIV/SIV pathogenesis by serving as a reservoir for viral persistence in brain and other tissues. Infected macrophages have been detected in brain early after infection, but macrophage-tropic viruses are rarely isolated until late-stage infection. Little is known about early variants that establish persistent infection in brain. Here, we characterize a unique macrophage-tropic SIV envelope glycoprotein (Env) variant from two weeks post-infection in blood of an SIVmac251-infected macaque that is closely related to sequences in brain from animals with neurological disease. SIVmac251 clones expressing this Env are highly fusogenic, and replicate efficiently in T cells and macrophages. N173 and N481 were identified as novel determinants of macrophage tropism and neutralization sensitivity. These results imply that macrophage-tropic SIV capable of establishing viral reservoirs in brain can be present in blood during early infection. Furthermore, these SIVmac251 clones will be useful for studies on pathogenesis, eradication, and vaccines. •We characterize a macrophage-tropic SIVmac251 variant in blood from early infection.•This Env variant is similar to brain variants from animals with neurological disease.•These sequences are also related to those in non-brain tissues such as lymph nodes.•SIVmac251 clones expressing these variants replicate in both T-cells and macrophages.•These SIVmac251 clones can be used to study pathogenesis, eradication, and vaccines

A Comparison of Urolithiasis in the Presence and Absence of Microscopic Hematuria in the Emergency Department

Introduction: Urolithiasis is a common medical condition that accounts for a large number of emergency department (ED) visits each year and contributes significantly to annual healthcare costs. Urinalysis is an important screening test for patients presenting with symptoms suspicious for urolithiasis. At present there is a paucity of medical literature examining the characteristics of ureteral stones in patients who have microscopic hematuria on urinalysis versus those who do not. The purpose of this study was to examine mean ureteral stone size and its relationship to the incidence of clinically significant hydronephrosis in patients with and without microscopic hematuria.Methods: This is a retrospective chart review of patient visits to a single, tertiary academic medical center ED between July 1, 2008, and August 1, 2013, of patients who underwent non-contrast computed tomography of the abdomen and pelvis and urinalysis. For patient visits meeting inclusion criteria, we compared mean stone size and the rate of moderate-to-severe hydronephrosis found on imaging in patients with and without microscopic hematuria on urinalysis.Results: Out of a total of 2,370 patient visits 393 (16.6%) met inclusion criteria. Of those, 321 (82%) had microscopic hematuria present on urinalysis. Patient visits without microscopic hematuria had a higher rate of moderate-to-severe hydronephrosis (42%), when compared to patients with microscopic hematuria present (25%, p=.005). Mean ureteral stone size among patient visits without microscopic hematuria was 5.7 mm; it was 4.7 mm for those patients with microscopic hematuria (p=.09). For ureteral stones 5 mm or larger, the incidence of moderate-to-severe hydronephrosis was 49%, whereas for ureteral calculi less than 5 mm in size, the incidence of moderate-to-severe hydronephrosis was 14% (p &lt; 0.0001).Conclusion: Patients visiting the ED with single-stone ureterolithiasis without microscopic hematuria on urinalysis could be at increased risk of having moderate-to-severe hydronephrosis compared to similar patients presenting with microscopic hematuria on urinalysis. Although the presence of hematuria on urinalysis is a moderately sensitive screening test for urolithiasis, these results suggest patients without hematuria tend to have more clinically significant ureteral calculi, making their detection more important. Clinicians should maintain a high index of suspicion for urolithiasis, even in the absence of hematuria, since ureteral stones in these patients were found to be associated with a higher incidence of obstructive uropathy

Bioinformatic Prediction Programs Underestimate the Frequency of CXCR4 Usage by R5X4 HIV Type 1 in Brain and Other Tissues

Human immunodeficiency virus (HIV-1) variants in brain primarily use CCR5 for entry into macrophages and microglia, but dual-tropic (R5X4) HIV-1 has been detected in brain and cerebral spinal fluid (CSF) of some patients with HIV-associated dementia (HAD). Here, we sequenced the gp120 coding region of nine full-length dual-tropic (R5X4) env genes cloned directly from autopsy brain and spleen tissue from an AIDS patient with severe HAD. We then compiled a dataset of 30 unique clade B R5X4 Env V3 sequences from this subject and 16 additional patients (n = 4 brain and 26 lymphoid/blood) and used it to compare the ability of six bioinformatic algorithms to correctly predict CXCR4 usage in R5X4 Envs. Only one program (SVMgeno2pheno) correctly predicted the ability of R5X4 Envs in this dataset to use CXCR4 with 90% accuracy (n = 27/30 predicted to use CXCR4). The PSSMSINSI, Random Forest, and SVMgenomiac programs and the commonly used charge rule correctly predicted CXCR4 usage with >50% accuracy (22/30, 16/30, 19/30, and 25/30, respectively), while the PSSMX4R5 matrix and “11/25” rule correctly predicted CXCR4 usage in <50% of the R5X4 Envs (10/30 and 13/30, respectively). Two positions in the V3 loop (19 and 32) influenced coreceptor usage predictions of nine R5X4 Envs from patient MACS1 and a total of 12 Envs from the dataset (40% of unique V3 sequences). These results demonstrate that most predictive algorithms underestimate the frequency of R5X4 HIV-1 in brain and other tissues. SVMgeno2pheno is the most accurate predictor of CXCR4 usage by R5X4 HIV-1

Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5

Topical agents, such as microbicides, that can protect against human immunodeficiency virus (HIV) transmission are urgently needed. Using a chimeric simian/human immunodeficiency virus (SHIV SF162), which is tropic for the chemokine receptor CCR5, we report that topical application of high doses of PSC-RANTES, an amino terminus-modified analog of the chemokine RANTES, provided potent protection against vaginal challenge in rhesus macaques. These experimental findings have potentially important implications for understanding vaginal transmission of HIV and the design of strategies for its prevention