Genome-wide association study of HIV-associated neurocognitive disorder (HAND): A CHARTER group study.

HIV-associated neurocognitive disorder (HAND) often complicates HIV infection despite combination antiretroviral therapy (ART) and may be influenced by host genomics. We performed a genome-wide association study (GWAS) of HAND in 1,050 CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) Study participants. All participants underwent standardized, comprehensive neurocognitive, and neuromedical assessments to determine if they had cognitive impairment as assessed by the Global Deficit Score (GDS), and individuals with comorbidities that could confound diagnosis of HAND were excluded. Neurocognitive outcomes included GDS-defined neurocognitive impairment (NCI; binary GDS, 366 cases with GDS ≥ 0.5 and 684 controls with GDS < 0.5, and GDS as a continuous variable) and Frascati HAND definitions that incorporate assessment of functional impairment by self-report and performance-based criteria. Genotype data were obtained using the Affymetrix Human SNP Array 6.0 platform. Multivariable logistic or linear regression-based association tests were performed for GDS-defined NCI and HAND. GWAS results did not reveal SNPs meeting the genome-wide significance threshold (5.0 × 10-8 ) for GDS-defined NCI or HAND. For binary GDS, the most significant SNPs were rs6542826 (P = 8.1 × 10-7 ) and rs11681615 (1.2 × 10-6 ), both located on chromosome 2 in SH3RF3. The most significant SNP for continuous GDS was rs11157436 (P = 1.3 × 10-7 ) on chromosome 14 in the T-cell-receptor alpha locus; three other SNPs in this gene were also associated with binary GDS (P ≤ 2.9 × 10-6 ). This GWAS, conducted among ART-era participants from a single cohort with robust neurological phenotyping, suggests roles for several biologically plausible loci in HAND that deserve further exploration. © 2017 Wiley Periodicals, Inc.

Cerebrospinal fluid cell-free mitochondrial DNA is associated with HIV replication, iron transport, and mild HIV-associated neurocognitive impairment.

BACKGROUND: Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. METHODS: We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure (GDS ≥ 0.5, impaired vs. GDS < 0.5, unimpaired). CSF, clinical, and biomarker data from the earliest available time point were analyzed. Cell-free mtDNA associations with CSF inflammation and iron-related biomarkers [CXCL10, IL-6, IL-8, TNF-a, transferrin (TF), ceruloplasmin (CP), and vascular endothelial growth factor (VEGF)], VL, and GDS were evaluated by multivariable regression. RESULTS: CSF cell-free mtDNA levels were significantly lower in participants with undetectable (vs. detectable) VL in either plasma (p < 0.001) or CSF (p < 0.001) and in those on antiretroviral therapy (ART; p < 0.001). Participants on ART with undetectable VL in both CSF and plasma had lower mtDNA levels than those with detectable VL in both compartments (p = 0.001). Higher mtDNA levels were observed in participants in the highest vs. lowest tertile (T3 vs. T1) of CSF CXCL10 (T3 vs. T1, p < 0.001) and TNF-a (T3 vs. T1, p < 0.05) in unadjusted analyses. MtDNA levels also correlated with CSF leukocyte count. After adjusting for CSF leukocyte count and VL, mtDNA levels were also associated with other inflammation- and iron-related biomarkers in CSF, including TF (T3 vs. T1, p < 0.05) and CP (T3 vs. T1, p < 0.05). With additional correction for ART use, mtDNA was also negatively associated with CSF VEGF (p < 0.05) and IL-6 (p = 0.05). We observed no associations of CSF mtDNA levels with age or GDS-defined NC impairment. CONCLUSIONS: CSF cell-free mtDNA levels were associated with HIV RNA and ART status, as well as with biomarkers of iron transport and VEGF, a growth factor with known effects on mitochondrial integrity and autophagy. CSF mtDNA may be a biomarker of iron dysregulation and/or neuroinflammation during HIV infection.

Genetic variation in iron metabolism is associated with neuropathic pain and pain severity in HIV-infected patients on antiretroviral therapy.

HIV sensory neuropathy and distal neuropathic pain (DNP) are common, disabling complications associated with combination antiretroviral therapy (cART). We previously associated iron-regulatory genetic polymorphisms with a reduced risk of HIV sensory neuropathy during more neurotoxic types of cART. We here evaluated the impact of polymorphisms in 19 iron-regulatory genes on DNP in 560 HIV-infected subjects from a prospective, observational study, who underwent neurological examinations to ascertain peripheral neuropathy and structured interviews to ascertain DNP. Genotype-DNP associations were explored by logistic regression and permutation-based analytical methods. Among 559 evaluable subjects, 331 (59%) developed HIV-SN, and 168 (30%) reported DNP. Fifteen polymorphisms in 8 genes (p<0.05) and 5 variants in 4 genes (p<0.01) were nominally associated with DNP: polymorphisms in TF, TFRC, BMP6, ACO1, SLC11A2, and FXN conferred reduced risk (adjusted odds ratios [ORs] ranging from 0.2 to 0.7, all p<0.05); other variants in TF, CP, ACO1, BMP6, and B2M conferred increased risk (ORs ranging from 1.3 to 3.1, all p<0.05). Risks associated with some variants were statistically significant either in black or white subgroups but were consistent in direction. ACO1 rs2026739 remained significantly associated with DNP in whites (permutation p<0.0001) after correction for multiple tests. Several of the same iron-regulatory-gene polymorphisms, including ACO1 rs2026739, were also associated with severity of DNP (all p<0.05). Common polymorphisms in iron-management genes are associated with DNP and with DNP severity in HIV-infected persons receiving cART. Consistent risk estimates across population subgroups and persistence of the ACO1 rs2026739 association after adjustment for multiple testing suggest that genetic variation in iron-regulation and transport modulates susceptibility to DNP.

Mitochondrial DNA variation and HIV-associated sensory neuropathy in CHARTER.

HIV-associated sensory neuropathy remains an important complication of combination antiretroviral therapy and HIV infection. Mitochondrial DNA haplogroups and single nucleotide polymorphisms (SNPs) have previously been associated with symptomatic neuropathy in clinical trial participants. We examined associations between mitochondrial DNA variation and HIV-associated sensory neuropathy in CNS HIV Antiretroviral Therapy Effects Research (CHARTER). CHARTER is a USA-based longitudinal observational study of HIV-infected adults who underwent a structured interview and standardized examination. HIV-associated sensory neuropathy was determined by trained examiners as ≥1 sign (diminished vibratory and sharp-dull discrimination or ankle reflexes) bilaterally. Mitochondrial DNA sequencing was performed and haplogroups were assigned by published algorithms. Multivariable logistic regression of associations between mitochondrial DNA SNPs, haplogroups, and HIV-associated sensory neuropathy were performed. In analyses of associations of each mitochondrial DNA SNP with HIV-associated sensory neuropathy, the two most significant SNPs were at positions A12810G [odds ratio (95 % confidence interval) = 0.27 (0.11-0.65); p = 0.004] and T489C [odds ratio (95 % confidence interval) = 0.41 (0.21-0.80); p = 0.009]. These synonymous changes are known to define African haplogroup L1c and European haplogroup J, respectively. Both haplogroups were associated with decreased prevalence of HIV-associated sensory neuropathy compared with all other haplogroups [odds ratio (95 % confidence interval) = 0.29 (0.12-0.71); p = 0.007 and odds ratio (95 % confidence interval) = 0.42 (0.18-1.0); p = 0.05, respectively]. In conclusion, in this cohort of mostly combination antiretroviral therapy-treated subjects, two common mitochondrial DNA SNPs and their corresponding haplogroups were associated with a markedly decreased prevalence of HIV-associated sensory neuropathy.

Continued high prevalence and adverse clinical impact of human immunodeficiency virus-associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER Study.

OBJECTIVE: To provide updated estimates of the prevalence and clinical impact of human immunodeficiency virus-associated sensory neuropathy (HIV-SN) and neuropathic pain due to HIV-SN in the combination antiretroviral therapy (CART) era. DESIGN: Prospective, cross-sectional analysis. Clinical correlates for HIV-SN and neuropathic pain, including age, exposure to CART, CD4 levels, plasma viral load, hepatitis C virus infection, and alcohol use disorders, were evaluated in univariate and multivariate models. SETTING: Six US academic medical centers. PATIENTS: One thousand five hundred thirty-nine HIV-infected individuals enrolled in the CNS (Central Nervous System) HIV Anti-Retroviral Therapy Effects Research study. MAIN OUTCOME MEASURES: The presence of HIV-SN, defined by 1 or more clinical signs (diminished vibration or sharp sensation in the legs and feet; reduced ankle reflexes) in a distal, symmetrical pattern. Neuropathic pain was defined as aching, stabbing, or burning in a similar distribution. The effect on quality of life was assessed with the Medical Outcomes Study HIV Health Survey. RESULTS: We found HIV-SN in 881 participants. Of these, 38.0% reported neuropathic pain. Neuropathic pain was significantly associated with disability in daily activities, unemployment, and reduced quality of life. Risk factors for HIV-SN after adjustment were advancing age (odds ratio, 2.1 [95% confidence interval, 1.8-2.5] per 10 years), lower CD4 nadir (1.2 [1.1-1.2] per 100-cell decrease), current CART use (1.6 [1.3-2.8]), and past "D-drug" use (specific dideoxynucleoside analogue antiretrovirals) (2.0 [1.3-2.6]). Risk factors for neuropathic pain were past D-drug use and higher CD4 nadir. CONCLUSIONS: Neuropathic pain and HIV-SN remain prevalent, causing substantial disability and reduced quality of life even with successful CART. The clinical correlates of HIV-SN have changed with the evolution of treatment. These findings argue for redoubled efforts to determine HIV-SN pathogenesis and the development of symptomatic and neuroregenerative therapies.

Role of metabolic syndrome components in HIV-associated sensory neuropathy.

OBJECTIVES: Sensory neuropathy is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy. Metabolic syndrome (MetS), a cluster of risk factors for atherosclerosis and microvascular disease, is associated with sensory neuropathy in HIV-uninfected (HIV-negative) persons. We examined whether MetS or its components predispose individuals to HIV-associated sensory neuropathy (HIV-SN). DESIGN: From a prospective multicenter cohort of 1556 HIV-positive patients, a subgroup (n = 130) with fasting laboratory tests and sensory neuropathy assessment was selected. METHODS: Sensory neuropathy was defined by symmetrically decreased reflexes or sensation loss in the legs. MetS was defined by presence of at least three risk factors: mean arterial pressure of at least 100 mmHg; triglycerides (TRGs) of at least 150 mg/dl and high-density lipoprotein cholesterol of less than 40 mg/dl for male patients, less than 50 mg/dl for female patients; body mass index of more than 25 kg/m; plasma glucose (GLU) of at least 100 mg/dl and self-reported diabetes mellitus type 2. Multivariate logistic regression examined the association between HIV-SN and MetS. RESULTS: After controlling for HIV-SN risk factors such as age, CD4 current, length of HIV infection, use of dideoxynucleoside reverse transcriptase inhibitors and protease inhibitors, MetS was not associated with HIV-SN (P = 0.72). However, when each MetS component was assessed, elevated TRG was a significant risk factor for HIV-SN. From the larger cohort, both diabetes mellitus type 2 (odds ratio = 1.4, P < 0.01) and elevated TRG (odds ratio = 1.4, P = 0.01) were risk factors for HIV-SN. CONCLUSION: The risk of HIV-SN was increased for diabetes mellitus type 2 and elevated TRG but not for other MetS components. Both increase the risk of sensory neuropathy in HIV-populations, but the mechanism(s) remains unclear.

Role of metabolic syndrome components in human immunodeficiency virus-associated stroke.

Metabolic syndrome (MetS) is a cluster of risk factors, including elevated mean arterial pressure (MAP), atherogenic dyslipidemia (elevated triglycerides [TRG]), abdominal obesity (increased body mass index [BMI]), glucose intolerance (elevated glucose [GLU]), and prothrombotic/inflammatory state (increases in uric acid [UA]), that are associated with increased risk of cerebrovascular disease. We studied if an association existed between MetS components and human immunodeficiency virus (HIV)-associated cryptogenic strokes-those not caused by HIV complications, endocarditis, or stimulant abuse. We performed a retrospective case-control study. Eleven cryptogenic strokes were identified from 2346 HIV-infected (HIV+) participants. Each case was matched by age, sex, and date of stroke diagnosis to five HIV+ controls without stroke. Nonparametric stratified Wilcoxon ranked sum tests with subsequent mixed effect logistic regression determined the influence of each MetS component on HIV-associated cryptogenic stroke. Although each MetS component appeared higher for HIV+ cases with cryptogenic strokes than HIV+ controls, only MAP (odds ratio [OR] = 5.70, 95% confidence interval [CI] = 1.15-28.3) and UA (OR = 1.88, 95% CI = 1.06-3.32) were statistically different. A significantly higher percentage of HIV-associated cryptogenic stroke cases met criteria for MetS (4/11 = 36%) compared to HIV+ controls (6/55 = 11%). This observational study suggests a possible role for MetS components in HIV+ cryptogenic stroke cases. Although MetS is defined as a constellation of disorders, elevated hypertension and hyperuricemia may be involved in stroke pathogenesis. Reducing MetS component levels in HIV+ patients could therefore protect them from subsequent stroke.

Cognitive functioning during highly active antiretroviral therapy interruption in human immunodeficiency virus type 1 infection.

Although no longer considered therapeutically beneficial, antiretroviral treatment interruptions (TIs) still occur frequently among patients with human immunodeficiency virus (HIV) infection for a variety of reasons. TIs typically result in viral rebound and worsening immunosuppression, which in turn are risk factors for neurocognitive decline and dementia. We sought to determine the extent of neurocognitive risk with TIs and subsequent reintroduction of highly active antiretroviral therapy (HAART) by using a comprehensive, sensitive neuropsychological assessment and by concurrently determining changes in plasma and cerebrospinal fluid (CSF) viral load and CD4 counts. Prospective, serial, clinical evaluations including neuropsychological (NP) testing and measurement of plasma HIV RNA and CD4 count and mood state were performed on HIV-1-infected individuals (N=11) at three time points: (1) prior to a TI, while on HAART; (2) after TIs averaging 6 months; and (3) after reinitiating HAART therapy. During TI, plasma HIV RNA increased and CD4 counts declined significantly, but NP performance did not change. Following reinitiation of HAART, viral loads fell below pre-TI levels, and CD4 counts rose. Improved viral suppression and immune restoration with reinitiation of HAART resulted in significant improvement in neurocognitive performance. No changes on comprehensive questionnaires of mood state were observed in relation to TI.NP performance and mood state remained stable during TIs despite worsened viral loads and CD4 counts. Because "practice effects" are generally greatest between the first and second NP testing sessions, improvement at the third, post-TI time point was unlikely to be accounted for by practice. TIs of up to 6 months appear to be neurocognitively and psychiatrically safe for most patients.

Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-beta) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.

Functional characterization of the genomic promoter of borna disease virus (BDV): implications of 3'-terminal sequence heterogeneity for BDV persistence.

Borna disease virus (BDV) is an enveloped virus with a genome organization characteristic of Mononegavirales. However, based on its unique features, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. We have described the establishment of a reverse genetics system for the rescue of BDV RNA analogues, or minigenomes, that is based on the use of polymerase I/polymerase II. Using this BDV minigenome rescue system, we have examined the functional implications of the reported sequence heterogeneity found at the 5' and 3' termini of the BDV genome and also defined the minimal BDV genomic promoter within the 3'-terminal 25 nucleotides. Our results suggest that the accumulation of RNA genome species containing truncations of one to three nucleotides at their 3' termini may contribute to modulate BDV RNA replication and gene expression during long-term persistence.

A reverse genetics system for Borna disease virus.

Borna disease virus (BDV) is an enveloped virus. Its non-segmented, negative-stranded RNA genome has the coding capability for six main polypeptides and has an organization characteristic of members of the order Mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the establishment of a reverse genetics system for BDV is described. Intracellular synthesis of a BDV RNA analogue or minigenome (MG) from a plasmid was driven by RNA polymerase I. Co-transfection with plasmids expressing the BDV polymerase (L), nucleoprotein (N) and phosphoprotein (P) under the control of RNA polymerase II allowed for BDV MG replication and expression. This process depended on a delicate N:P ratio, whereas the L:P ratio was less critical. Two isoforms of N, Np40 and Np38, are present in BDV-infected cells but only Np40 was strictly required for virus polymerase activity. BDV p10 polypeptide encoded by the P gene exhibited a strong inhibitory effect on BDV MG expression.