Expression of CHRFAM7A and CHRNA7 in neuronal cells and post-mortem brain of HIV-infected patients: Considerations for HIV-Associated Neurocognitive Disorder

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Despite the recent advances in antiretroviral therapy, HIV-1 remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death, neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation pattern in various gp120IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant down-regulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expression in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.

Keywords: gp120, HIV, HAND, acetylcholine receptor, CHRNA7, CHRFAM7A



The human immunodeficiency virus type 1 (HIV-1) is considered one of the principal pandemics of the twenty-first century with approximately 34 million of subjects infected globally (Joint United Nations Programme on HIV/AIDS (UNAIDS 2013). In addition to developing acquired immunodeficiency syndrome (AIDS), infected individuals may also develop neurological complications known as HIV-associated neurocognitive disorders (HAND). HAND includes asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV-associated dementia (HAD) (Antinori et al. 2007). HAD results in disabling cognitive impairment accompanied by motor dysfunction, speech problems, and overt behavioral changes (González-Scarano and Martín-García 2005; Clifford and Ances 2013). Although the incidence of HAD has decreased (Bhaskaran et al. 2008), the prevalence of HAND, mostly of the milder forms of neurocognitive impairment (ANI and MND), could be as high as 50% of patients (Sacktor et al. 2002; Cysique et al. 2004; Heaton et al. 2011). Moreover, the high prevalence of HAND occurs despite administration of combined antiretroviral therapy (cART) (Mothobi and Brew 2012). For instance, under cART, HAND persists despite systemic or brain viral load reduction or control (Cysique and Brew 2011; Koneru et al. 2014).

HIV is unable to infect neurons due to their lack of primary CD4 receptors, however, neuronal expression of both CCR5 and CXCR4 secondary receptors could allow viral interactions (Hesselgesser et al. 1997). Several hypotheses have emerged to explain the cause of HAND including the neurotoxic properties of viral proteins and the severe uncontrolled chronic neuroinflammation (Kong et al. 1996; Heaton et al. 2011). Particularly, the HIV-1 viral envelope protein gp120 has been reported to have various neurotoxic properties in vitro and in vivo including the inhibition of adult neural progenitor cells proliferation, neuronal damage and induction of apoptosis, and cell death of human neuronal cells (Toggas et al. 1994; Meucci and Miller 1996; Hesselgesser et al. 1998; Jana and Pahan 2004; Bardi et al. 2006; Okamoto et al. 2007; Ballester et al. 2012). Moreover, the severity of brain damage correlates with gp120 levels (Desai et al. 2013).

The alpha7 nicotinic acetylcholine receptor (α7) is one of the most common receptors expressed in the mammalian brain (Dani and Lester 2001). The α7 subunit is encoded by the CHRNA7 gene in chromosome 15, and is composed of 10 exons (Gault et al. 1998). Interestingly, the CHRNA7 has a counterpart gene named CHRFAM7A (Gault et al. 1998). The CHRFAM7A gene product, dupα7, exerts a regulatory/inhibitory role on the α7 ion channel activity and expression (de Lucas-Cerrillo et al. 2011; Araud et al. 2011), although a recent work has challenged these results showing that dupα7 and α7 can form functional heteropentamers with altered responses to choline and varenicline (Wang et al. 2014). This may be due to differences in the expression system used that could influence ion channel functionality and assembly –the first study used oocytes while the most recent used Neuro2a cells–, and the use of α7's chaperone RIC-3 in Neuro2a cells but not in oocytes. For a comprehensive review about dupα7 refer to (Costantini et al. 2014). Notwithstanding, although the α7 has been amply studied in CNS, very little is known about its role in the neuropathology of HIV infection. We recently demonstrated that gp120IIIB induces a functional α7 upregulation in neuronal cells and that the expression of gp120IIIB in the brain of a transgenic mouse model also induces the overexpression of α7 in the brain, particularly in the striatum, basal ganglia's primary input (Ballester et al. 2012). Moreover, we found that the activation of upregulated α7 in these neuronal cells induces cell death in a calcium-dependent manner (Ballester et al. 2012). In light of the possible role of α7 in the HIV neuropathogenesis, we evaluated the mRNA expression patterns of CHRNA7, CHRFAM7A and the expression ratio CHRNA7:CHRFAM7A upon gp120IIIB application in a human neuronal cell line and in post-mortem brain samples from HIV-infected patients expressing different severity stages of neurocognitive impairment.

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Materials and methods


All reagents were purchased from Sigma Aldrich unless otherwise specified.

Cell culture and treatments

SH-SY5Y neuronal cell line was obtained from ATCC (Manassas, VA). Cells were incubated at 37 °C with 5% CO2 in DMEM/F-12 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1.2 g of NaHCO3. Cultures were performed in 12-well plates followed by treatments with gp120IIIB (Fitzgerald Industries International, Concord, MA) at 0.0015, 0.015, 0.15, 1.5 or 15 nM for the indicated time. For time-dependent experiments, the concentration of gp120IIIB was 0.15 nM. The CXCR4 antagonist, AMD3100 (EMD Chemicals, Inc., Gibbstown, NJ), was used at 1 μM and added 10 min prior to gp120IIIB application. The range of gp120 concentrations tested here were based on gp120 quantification studies using plasma, serum and tissues from HIV-infected subjects (Gilbert et al. 1991; Oh et al. 1992; Santosuosso et al. 2009; Rychert et al. 2010). To our knowledge there is no quantification studies to determine gp120 in the brain or cerebrospinal fluid (CSF). However, there is a robust body of evidence demonstrating that indeed gp120 is present in the central nervous system and CSF, even though no evidence of quantification is available in the literature (Buzy et al. 1989; Rolfs and Schumacher 1990; Ruta et al. 1998; Cashion et al. 1999; Jones et al. 2000; Ohagen et al. 2003; Pillai et al. 2006). Moreover, the existence of anti-gp120 antibodies in the CNS unequivocally attests to its presence (Lucey et al. 1993; Di Stefano et al. 1996; Trujillo et al. 1996).

RNA extraction and quantitative RT-PCR assay

In SH-SY5Y neuronal cells, total RNA samples were extracted using TriZol Reagent (Invitrogen Corporation, Carlsbad, CA). To eliminate possible genomic contamination, extracted RNA was treated with DNase using the Ambion DNA free kit (Ambion, Austin, TX). Quantification of total RNA was performed using a Nanodrop system (Thermo Scientific, Waltham, MA). The cDNA synthesis was carried out using 0.75 μg of total RNA with the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA) following the manufacturer's instructions. After optimization of the PCR conditions, real-time PCR experiments were performed using the iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA) in a Mastercycler® Ep Realplex Thermal Cycler (Eppendorf, NY). CHRNA7 and GAPDH primers where used at a final concentration of 400 nM, CHRFAM7A primers at 100 nM together with 100 ng of cDNA. Primers were designed using IDT Designer Software (Integrated DNA Technologies). The primers employed to amplify the genes of interest from cells and tissue samples were the following: CHRNA7 forward, 5′-GCTCCGGGACTCAACATG-3′; reverse, 5′-GGGATTGTAGTTCTTGACCAGC-3′; CHRFAM7A forward, 5′-CCGAAGTTACTGGCCTCTATC-3′ reverse, 5′-CTGAGTCGTGTAGATAAGCTCTC-3′, and for GAPDH: forward, 5′-GCTCTCTGCTCCTCCTGTTC-3′, reverse, 5′-GACTCCGACCTTCACCTTCC-3′. All primers were used with an annealing temperature of 55 °C.

Tissue processing and RNA extraction

Post-mortem brain tissues from HIV-infected patients were obtained from the Texas NeuroAIDS Research Center (IRB#: 98-402), California NeuroAIDS Tissue Network (IRBs#: 00000353, 00000354, 00000355, and 000002758), and UCLA National Neurological AIDS Bank (IRB#: 10000525) Tissue samples were pulverized in liquid nitrogen under RNAse free conditions. RNA extraction was performed using TriZol reagent (Invitrogen) following manufacturer's instructions. The RNA integrity was assayed in 1% electrophoresis agarose gel. Samples were processed for qRT-PCR as described above.

Statistical analyses

To evaluate the statistical significance of changes in expression levels of CHRNA7 and CHRFAM7A in neuronal cells we used One-way ANOVA followed by Holm-Sidak's multiple comparison test which allowed corrections for multiple comparisons with a fixed alpha value (0.05). Spearman correlation was used to identify correlations between CHRFAM7A and CHRNA7 expression levels in neuronal cells. The detected outliers were excluded from analysis. Statistical analysis was conducted using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA,

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gp120IIIB promotes the downregulation of CHRFAM7A in neuronal cells

Neuronal cells were exposed to various concentrations of gp120IIIB including those within the pathophysiological range quantified in HIV-infected patients (0.0015, 0.015 and 0.15 nM) (Gilbert et al. 1991; Oh et al. 1992; Santosuosso et al. 2009; Rychert et al. 2010). Measurements of CHRNA7 and CHRFAM7A levels after addition of pathophysiological relevant gp120IIIB concentrations show that the CHRFAM7A was downregulated in a dose-dependent manner, and that the expression of CHRNA7 was induced (Fig. 1a). Noteworthy is that this effect in CHRFAM7A expression levels is sustained even when supraphysiological concentrations of gp120IIIB were used (15 nM). Further evaluation shows that CHRNA7:CHRFAM7A expression ratios increase with the gp120IIIB treatment (Fig. 1b).

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Fig. 1

gp120IIIB induces the downregulation of CHRFAM7A in neuronal cells. a Neuronal cells were incubated with gp120IIIB 0.0015, 0.015, 0.15, 1.5 and 15.0 nM for 12 hours. A downregulation of CHRFAM7A was observed under pathophysiological (0.015 and 0.15 nM) and supraphysiological doses (15 nM). b CHRNA7:CHRFAM7A expression ratio shows a significant increase under pathophysiological and supraphysiological conditions as compared to control. In panel a and b, results were normalized to control. *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001. Statistical analysis: One-way ANOVA followed by Holm-Sidak's multiple comparison tests, error bars represents SEM. For all panels n = 4 independent experiments.

A pathophysiological dose of gp120IIIB time-dependently dysregulates CHRNA7 and CHRFAM7A expression in neuronal cells

CXCR4 is a coreceptor employed by HIV to infect immune cells and is expressed by neurons (Hesselgesser et al. 1997). Neuronal cells exposed to gp120IIIB (0.15 nM) at different time points showed that the α7 gene, CHRNA7, was upregulated after 12 hours post gp120IIIB exposure whereas CHRFAM7A downregulation initiated as early as 15 minutes post gp120IIIB application, and lasts 24 hours (Fig. 2a). Moreover, a ratio analysis demonstrates an early increase in the CHRNA7:CHRFAM7A expression (Fig 2b).

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Fig. 2

Time-dependent responses of CHRNA7 and CHRFAM7A in neuronal cells exposed to HIV-1-gp120IIIB. a Neuronal cells were incubated with gp120IIIB (0.15 nM) at various time points. As compared to untreated control cells, downregulation of CHRFAM7A was observed at all-time points while CHRNA7 was upregulated after 12 hours of gp120IIIB application. b CHRNA7:CHRFAM7A expression ratio showed a significant increase after 15 min, 12h, and 24h post gp120IIIB application. Results were normalized and compared to control cells. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. n = 4 independent experiments. Statistical analysis: One-way ANOVA followed by Holm-Sidak's multiple comparison test.

A CXCR4 antagonist abrogates the gp120IIIB-induced dysregulation of CHRNA7

To determine whether the CHRNA7 and CHRFAM7A dysregulation depends on CXCR4 stimulation, an antagonist (AMD3100) was applied prior to gp120IIIB addition. Our results show that CXCR4 blockade abrogates gp120IIIB-induced upregulation of CHRNA7 (Fig. 3). Unexpectedly, CHRFAM7A was downregulated by AMD3100 in the absence of gp120IIIB (Fig 3a).

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Fig. 3

Dysregulation of the CHRFAM7A:CHRNA7 ratio by gp120IIIB is dependent on CXCR4-gp120 interaction. Neuronal cells were incubated with 0.15 nM gp120IIIB for 12 hours with and without a pre-incubation of AMD3100. The CHRNA7 upregulation induced by gp120IIIB was obliterated by AMD3100 treatment. AMD3100 treatment alone downregulates the CHRFAM7A. Results were normalized and compared to control cells. *P ≤ 0.05, **P ≤ 0.01. n = 3 independent experiments. Statistical analysis: One way ANOVA followed by a Holm-Sidak's multiple comparison tests.

The CHRNA7 and CHRFAM7A expression levels in the basal ganglia of HIV-infected subjects

It is known that the basal ganglia is an area of the brain that is severely affected in HIV-infected patients (Woods et al. 2009) and contain some of the brain's highest viral load (Kure et al. 1990). We recently found that the CHRNA7 gene product, α7, is upregulated in the striatum (a component of the basal ganglia) of mice expressing gp120IIIB in the brain (Ballester et al. 2012). Here we examined CHRNA7, CHRFAM7A, and CHRNA7:CHRFAM7A levels in the basal ganglia of HIV-infected post mortem basal ganglia samples representing different stages of neurological impairment. Table 1 summarizes the subject characteristics. Evaluation of CHRFAM7A and CHRNA7 genes in HIV+ patients shows that CHRNA7 is significantly expressed at higher levels than CHRFAM7A (Fig 4a), which is consistent with what we observed in the neuronal cells (Fig. 1a). The CHRNA7:CHRFAM7A ratio of the HIV+ group was increased in these patients (Fig. 4b). Examination of CHRNA7 levels in basal ganglia from HIV-infected subjects suffering from different stages of cognitive impairment showed no significant differences (Fig. 4c). In terms of CHRFAM7A, at first glance, patients with normal cognition are not different from HIV- (Fig. 4d) but detailed examination of the distribution of CHRFAM7A levels in normal cognition patients demonstrate two distinguishable groups identified as subgroups A and B (Fig. 4d). Evaluation of these groups revealed that subgroup A is upregulated while subgroup B is downregulated for CHRFAM7A expression (Fig. 4d). Furthermore, examination of CHRFAM7A levels in the MCMD group suggests that only HIV+ patients with low CHRFAM7A levels develop MCMD (Fig. 4e). Ratio analysis demonstrates no significant differences in the CHRNA7:CHRFAM7A expression ratio in the basal ganglia of these patients and a linear trend analysis showed a non-significant (P = 0.08) increment in CHRNA7:CHRFAM7A with increasing cognitive impairment severity (Fig. 4f).An external file that holds a picture, illustration, etc.
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Fig. 4

Dysregulation of the CHRFAM7A transcript in the basal ganglia of HIV+ individuals. a HIV-infected subjects exhibited significant higher levels of CHRNA7, HIV- (n = 5), HIV+ (n = 31). b CHRNA7:CHRFAM7A expression ratio appears higher in HIV-infected subjects HIV+ (n = 5), HIV- (n = 31). c Analysis of CHRNA7 mRNA levels in HIV-infected individuals. For control n = 5, normal cognition n = 13, MCMD n = 9, and HAD n = 11. d Two subgroups of HIV+ individuals with normal cognition exhibited different responses in CHRFAM7A regulation and are significantly different when compared to HIV- individuals. For control n = 5, normal cognition n = 16, subgroup A n = 7, and sub-group B n = 9. e No significant changes were detected in CHRFAM7A levels in MCMD or HAD suffering patients when compared to HIV- whereas normal cognition subgroups are significant different from HIV- subjects. For control n = 5, sub-group A n = 7, and sub-group B n = 9, MCMD n = 9, and HAD n = 13. f Analysis of variance followed by a linear trend test comparing all groups (P = 0.08). For control n = 5, normal cognition n = 10, MCMD n = 9, and HAD n = 12. For all panels, results were normalized to HIV- individuals. *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001.

Table 1

Subject characteristics



General characteristics

(n = 5)

Normal Cognition (n = 16)

MCMD (n = 9)

HAD (n =13)

* Age (years)

51.0 (48.0,51.0)

53.0 (46.0, 59.0)

44.0 (40.5, 58.5)

51.0 (44.5, 54.5)


5 male

15 male/1 female

9 male

13 male



RTV (1), 3TC (5), CBV (1), DRV (1), NVP (1), ABC (1), NA (6)

RTV (1), APV (1), CBV (2), 3TC (1), NA (4)

ZVD (1), FTC (1), RTV (1), APV (1), CBV (1), EFV (1), ABC (1), 3TC (1), NA (5)

Viral-immune profile

* CD4 cell count (cells/mm3)


171.0 (18.0, 352.0)

26.5 (7.8, 159.8)

247.0 (25.0, 365.0)

* Plasma HIV RNA (log 10)


4.6 (2.6, 5.2)

5.1 (4.3, 5.8)

4.3 (2.6, 5.1)

*Values presented as median (25th, 75th percentiles)

N/A = not applicable

NA = information not available

RTV = ritonavir (Norvir), 3TC = lamivudine (Epivir), CBV = zidovudine + lamivudine (Combivir), DRV = TMC-114, darunavir (Prezista), NVP = nevirapine (Viramune), ABC = abacavir (Ziagen), APV = amprenavir (Agenerase), CBV = zidovudine + lamivudine (Combivir), ZDV = AZT, zidovudine (Retrovir), FTC = emtricitabine (Coviracil; Emtriva), EFV = efavirenz (Sustiva).

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HIV-infected patients suffer from cognitive disorders associated with the infection. In a previous report we demonstrated that gp120IIIB is capable of inducing a functional upregulation of the α7 in neuronal cells and that this upregulation promotes cell death in a calcium-dependent manner (Ballester et al. 2012). In the current study, we expand these observations demonstrating that gp120IIIB induces the upregulation of the α7 gene CHRNA7 and the downregulation of its partial duplication, CHRFAM7A, in neuronal cells. The significant reduction in CHRFAM7A expression could imply that dupα7's dominant negative effect on α7's functionality may be concomitantly reduced, thus providing a modulatory/regulatory explanation for our previous observations (Ballester et al. 2012). Because of dupα7's dominant negative regulatory effect on α7, we evaluated the CHRNA7:CHRFAM7A ratio as indicative of the α7 functionality and found that gp120IIIB indeed does modify the ratio. In our study, we also used different concentrations of gp120IIIB to better understand its effects on CHRNA7 and CHRFAM7A expression. Our results show that the greatest observed reduction in CHRFAM7A expression together with a CHRNA7 induction occurs within the pathophysiological range of gp120IIIB documented for HIV-infected patients.

We also studied the kinetics of the gp120IIIB-induced CHRNA7 and CHRFAM7A dysregulation. Our results demonstrate that the gp120IIIB first induces a reduction in CHRFAM7A expression (15 min) followed by CHRNA7 induction (12 h), shedding light on the regulatory/modulatory mechanism behind the α7 upregulation which points to an early regulatory mechanism (before 15 min) by the CHRFAM7A gene. These results, together with our previous published observations demonstrating that the functional upregulation of α7 in neuronal cells promote cell death and that the α7 upregulation appears to be restricted to the basal ganglia (Ballester et al. 2012), are consistent with: (i) the neuronal apoptosis and cell death in the presence of gp120IIIB (×4), gp120 R5, and supernatants containing HIV-1 (Hesselgesser et al. 1998; Catani et al. 2000; Xu et al. 2004), (ii) the neuronal apoptosis identified in postmortem brain from adults and pediatric HIV-infected patients (Adle-Biassette et al. 1995; Gelbard et al. 1995), (iii) the basal ganglia neuronal density reduction in HIV-infected patients (Everall et al. 1995), (iv) autopsy studies of patients with HAD showing that the greatest burden of neuropathology is found in the basal ganglia (Brew et al. 1995), (v) the large accumulation of gp120 in humans' basal ganglia (Jones et al. 2000), and (vi) the neuronal dysfunction and cellular destruction identified in a transgenic mice expressing gp120 in the brain (Corboy et al. 1992; Toggas et al. 1994; Berrada et al. 1995).

Although neurons do not express CD4, they express functional CXCR4 and CCR5 coreceptors enabling gp120 to interact with them and activate signaling pathways leading to neuronal cell death (Kaul et al. 2005; Kaul et al. 2007). The role of CXCR4 in the gp120-mediated neurotoxicity can be direct, through the activation of neuronal receptors by gp120; or indirect through the stimulation of glial cells leading to release of neurotoxic factors (Ghafouri et al. 2006). The activation of CXCR4 by SDF-1α (CXCR4 endogenous agonist) or gp120 has been implicated in the mechanism for neuronal dysfunction during HAD (Hesselgesser et al. 1998; Zheng et al. 1999). Herein we report alterations in the gene expression of a cholinergic receptor and its partial duplication which are both amply distributed through the brain. Dysregulation of these genes under neuropathological settings is not new. For instance, the ratio of CHRNA7:CHRFAM7A mRNA levels is different in bipolar subjects when compared to unaffected controls (De Luca et al. 2006). Moreover, in vitro studies have demonstrated that two pro-inflammatory mediators characteristic of HIV-I infection, LPS and IL-1β, decrease CHRFAM7A expression leading to the suggestion that chronic pro-inflammatory responses might change the CHRFAM7A:CHRNA7 expression ratio (Benfante et al. 2011; van der Zanden et al. 2012). gp120IIIB is unable to promote CHRNA7:CHRFAM7A alterations in the presence of AMD3100, an antagonist of CXCR4, suggesting that the gp120IIIB-induced CHRNA7:CHRFAM7A dysregulation is CXCR4-dependent.

The cognitive impairments observed in HIV-infected subjects are the consequence of neurological alterations in the brain that compromise neural tracts resulting in significant damage and alterations of specific areas. The basal ganglia, which is one of the most severely affected areas in these patients (Berger and Nath 1997; Berger and Arendt 2000; Berger et al. 2000; von Giesen et al. 2001; Woods et al. 2009), contains cholinergic neurons and interneurons that express α7 (Azam et al. 2003; Bonsi et al. 2011). To better understand the clinical implications of our findings, analysis of CHRNA7 and CHRFAM7A genes were performed on post-mortem basal ganglia samples from HIV-infected individuals with different levels of neurological impairment severity. Our results demonstrate that regardless of the neurological impairment severity, the CHRNA7 was not significantly altered as compared to HIV- subjects. However, comparing the expression of the CHRNA7 and CHRFAM7A genes within HIV-infected patients reveals that the CHRNA7 expression is significantly increased in these patients (Fig. 4a). Interestingly, a closer look at the CHRFAM7A gene expression levels revealed two distinct populations within the normal cognition group: subgroup A and B. Of note, a significant increase was detected in the expression of the CHRFAM7A gene in subgroup A when compared to the HIV- group and subgroup B, and a significant reduction in the expression of CHRFAM7A in subgroup B was detected when compared to the HIV- group and subgroup A. In addition, comparing the CHRFAM7A expression in both subgroups reveals a statistically significant difference. A provocative hypothesis on the existence of these two discernible subgroups within the normal cognition group is that the patients exhibiting elevated levels of CHRFAM7A are less likely of suffering from HIV-associated cognition problems, and those with low levels of CHRFAM7A, within sub-group B, are more susceptible to develop neurological impairment as lower CHRFAM7A expression levels could imply a potentiation of the α7 receptor expression, increased calcium influx, and ultimate neuronal cell death (de Lucas-Cerrillo et al. 2011; Araud et al. 2011; Ballester et al. 2012). Because the tissues were collected before patients presenting ANI were distinguished from patients displaying normal cognition, subgroups A and B could comprise patients with either normal cognition or ANI. It is tempting to hypothesize that subgroup A comprise patients with normal cognition, and subgroup B comprise patients that presented ANI as patients presenting ANI are known to progress to more severe stages (Grant et al. 2014). Taking this into account, our results may imply that alterations in the expression of CHRNA7 and CHRFAM7A, or the CHRNA7:CHRFAM7A ratio might be detrimental to the cognitive performance of these patients.

In this study we tested the hypothesis that higher levels of neurological impairment could be associated with alterations in CHRNA7 or CHRFAM7A expression levels. Whether this dysregulation is responsible for the destruction of cholinergic neurons within the basal ganglia of HIV-infected patients remains to be determined. However, the available evidence points in that direction. For instance, (i) the basal ganglia of HIV-infected patients is compromised (Berger and Nath 1997; Berger et al. 2000; von Giesen et al. 2001; Woods et al. 2009), (ii) the α7 upregulation in the basal ganglia of transgenic mice expressing gp120 in the brain predispose this area to cell death events similar to what was detected in α7-upregulated neuronal cells (Ballester et al. 2012). Together, this evidence lead us to suggest that the alterations in the CHRNA7:CHRFAM7A expression might be implicated in the basal ganglia alterations observed in HIV-infected subjects with neurological impairments. This interpretation is supported by several lines of evidence showing that the motor dysfunction suffered by subjects, under pathological circumstances, involve compromised basal ganglia interneurons (Bonsi et al. 2011) reminiscent of MCMD-suffering patients.

In conclusion, we showed that gp120IIIB is capable of dysregulating the CHRNA7/CHRFAM7A expression in neuronal cells. Moreover, this dysregulation was detected in postmortem brain samples recovered from HIV-infected patients with different stages of HAND. The present study is limited in that the results from HIV+ patients basal ganglia may be hindered by the lack of statistical power to detect small changes in expression levels as statistically significant given the dispersion in the data; and that the normal cognition group may actually include HIV+ patients that presented asymptomatic neurocognitive impairment (ANI) because the tissues were collected before ANI was established as a classification category of HIV-induced neurocognitive disorders. Nevertheless, our results raises fundamental questions about the role of α7 and dupα7 in HIV-induced neurological disorders and warrants further statistically powered investigations using an increased number of brain samples from HIV-infected subjects under different stages of HAND. In addition, further studies aimed at exploring the CCR5 tropic gp120 (gp120JRFL) effects on α7 expression in neuronal cells are warranted. It would be interesting to determine whether CCR5 stimulation influences α7 expression as occurs with the CXCR4 tropic-specific gp120IIIB. In fact, it is known that activation of these G-protein coupled receptors produce similar signaling pathways (Davis et al. 1997; Lee et al. 2003) that, in the presence of gp120, could lead to death of neuronal cells (Catani et al. 2000), therefore, it would not be surprising that both gp120s produce similar responses.

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We are very grateful for Dr. Valerie Wojna's valuable suggestions and in depth review of this manuscript. We appreciate the National NeuroAIDS Tissue Consortium (NNTC) and the Data Coordinating Center (DCC) which is a project funded by the National Institute of Mental Health and the National Institute of Neurological Disorders and Stroke under the following grants: U24MH100928 (California NeuroAIDS Tissue Network), U24MH100930 (Texas NeuroAIDS Research Center), and U24MH100929 (UCLA National Neurological AIDS Bank). This research was supported by the National Institutes of Health-NINDS (2U54NS43011 to José A. Lasalde-Dominicci), the RISE Program from NIGMS (2R25GM061151 to Manuel Delgado-Vélez and Orestes Quesada) and MARC Program from NIGMS (5T34GM007821 to Orestes Quesada and Felix M. Ramos). Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD007587 (Carlos A. Báez-Pagán). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Conflict of interest: The authors declare that they have no competing interests.

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Jawahar Raina
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Pharmacological induction of CCL5 in vivo prevents gp120-mediated neuronal injury.

Pharmacological induction of CCL5 in vivo prevents gp120-mediated neuronal injury.


The human immunodeficiency virus (HIV) envelope protein gp120 promotes neuronal injury which is believed to cause HIV-associated neurocognitive disorders. Therefore, blocking the neurotoxic effect of gp120 may lead to alternative strategies to reduce the neurotoxic effect of HIV. In vitro, the neurotoxic effect of M-tropic gp120BaL is reduced by the chemokine CCL5, the natural ligand of CCR5 receptors. To determine whether CCL5 reduces the toxic effect of gp120BaL in vivo, animals were intrastriatally injected with lentiviral vectors overexpressing CCL5 prior to an intrastriatal injection of gp120BaL (400 ng). Neuronal injury was determined by silver staining, cleaved caspase-3 and TUNEL. Overexpression of CCL5 decreased gp120-mediated neuronal injury. CCL5 expression can be up-regulated by chronic morphine. Therefore, we examined whether morphine reduces the neurotoxic effect of gp120BaL. Rats stereotaxically injected with gp120BaL into the striatum received saline or chronic morphine for five days (10 mg/kg escalating to 30 mg/kg twice a day). Morphine-treated rats showed a decrease in all markers used to determine neuronal degeneration compared to saline-treated rats. The neuroprotective effect of morphine was significantly attenuated by expressing CCL5 shRNA. Our results suggest that compounds that increase the endogenous production of CCL5 may be used to reduce the pathogenesis of HIV-associated neurocognitive disorders.


CCL5-lentivirus; Caspase-3; IL-1β; Morphine withdrawal; Neurodegeneration; Neuroprotection

Jawahar Raina
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HIV-1 fitness cost associated with escape from the VRC01 class of CD4 binding site neutralizing antibodies.

HIV-1 fitness cost associated with escape from the VRC01 class of CD4 binding site neutralizing antibodies.


Broadly neutralizing antibodies (bNAbs) have been isolated from selected HIV-1-infected individuals and shown to bind to conserved sites on the envelope glycoprotein (Env). However, circulating plasma virus in these donors is usually resistant to autologous isolated bNAbs, indicating that during chronic infection, HIV-1 can escape from even broadly cross-reactive antibodies. Here, we evaluate if such viral escape is associated with an impairment of viral replication. Antibodies of the VRC01 class target the functionally conserved CD4 binding site and share a structural mode of gp120 recognition that includes mimicry of the CD4 receptor. We examined naturally occurring VRC01-sensitive and -resistant viral strains, as well as their mutated sensitive or resistant variants, and tested point mutations in the backbone of the VRC01-sensitive isolate YU2. In several cases, VRC01 resistance was associated with a reduced efficiency of CD4-mediated viral entry and diminished viral replication. Several mutations, alone or in combination, in the loop D or β23-V5 region of Env conferred a high level of resistance to VRC01 class antibodies, suggesting a preferred escape pathway. We further mapped the VRC01-induced escape pathway in vivo using Envs from donor 45, from whom antibody VRC01 was isolated. Initial escape mutations, including the addition of a key glycan, occurred in loop D and were associated with impaired viral replication; however, compensatory mutations restored full replicative fitness. These data demonstrate that escape from VRC01 class antibodies can diminish viral replicative fitness, but compensatory changes may explain the limited impact of neutralizing antibodies during the course of natural HIV-1 infection.


Some antibodies that arise during natural HIV-1 infection bind to conserved regions on the virus envelope glycoprotein and potently neutralize the majority of diverse HIV-1 strains. The VRC01 class of antibodies blocks the conserved CD4 receptor binding site interaction that is necessary for viral entry, raising the possibility that viral escape from antibody neutralization might exert detrimental effects on viral function. Here, we show that escape from VRC01 class antibodies can be associated with impaired viral entry and replication; however, during the course of natural infection, compensatory mutations restore the ability of the virus to replicate normally.

Jawahar Raina
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Structure and Dynamics of the Native HIV-1 Env Trimer

Structure and Dynamics of the Native HIV-1 Env Trimer


HIV-1/AIDS remains one of the worst pandemics in human history. Despite tremendous efforts, no effective vaccine has been found. Recent reports give new insights into the structure and dynamics of the HIV-1 Env trimer and renew hopes that a better understanding of Env will translate into new vaccine candidates and more-effective antiretroviral therapies.

Jawahar Raina
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An evolutionary role for HIV latency in enhancing viral transmission.

An evolutionary role for HIV latency in enhancing viral transmission.


HIV latency is the chief obstacle to eradicating HIV but is widely believed to be an evolutionary accident providing no lentiviral fitness advantage. However, findings of latency being "hardwired" into HIV's gene-regulatory circuitry appear inconsistent with latency being an evolutionary accident, given HIV's rapid mutation rate. Here, we propose that latency is an evolutionary "bet-hedging" strategy whose frequency has been optimized to maximize lentiviral transmission by reducing viral extinction during mucosal infections. The model quantitatively fits the available patient data, matches observations of high-frequency latency establishment in cell culture and primates, and generates two counterintuitive but testable predictions. The first prediction is that conventional CD8-depletion experiments in SIV-infected macaques increase latent cells more than viremia. The second prediction is that strains engineered to have higher replicative fitness—via reduced latency—will exhibit lower infectivity in animal-model mucosal inoculations. Therapeutically, the theory predicts treatment approaches that may substantially enhance "activate-and-kill" HIV-cure strategies.

Jawahar Raina
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Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5.

Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5.


Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120-CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues.


Bridging sheet; CCR5; CD4; Envelope; HIV; Macrophage; Tropism; V1/V2 stem

Jawahar Raina
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Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity.

Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity.


Adoptive transfer of CD8 T cells genetically engineered to express "chimeric antigen receptors" (CARs) represents a potential approach toward an HIV infection "functional cure" whereby durable virologic suppression is sustained after discontinuation of antiretroviral therapy. We describe a novel bispecific CAR in which a CD4 segment is linked to a single-chain variable fragment of the 17b human monoclonal antibody recognizing a highly conserved CD4-induced epitope on gp120 involved in coreceptor binding. We compared a standard CD4 CAR with CD4-17b CARs where the polypeptide linker between the CD4 and 17b moieties is sufficiently long (CD4-35-17b CAR) versus too short (CD4-10-17b) to permit simultaneous binding of the two moieties to a single gp120 subunit. When transduced into a peripheral blood mononuclear cell (PBMC) or T cells thereof, all three CD4-based CARs displayed specific functional activities against HIV-1 Env-expressing target cells, including stimulation of gamma interferon (IFN-γ) release, specific target cell killing, and suppression of HIV-1 pseudovirus production. In assays of spreading infection of PBMCs with genetically diverse HIV-1 primary isolates, the CD4-10-17b CAR displayed enhanced potency compared to the CD4 CAR whereas the CD4-35-17b CAR displayed diminished potency. Importantly, both CD4-17b CARs were devoid of a major undesired activity observed with the CD4 CAR, namely, rendering the transduced CD8(+) T cells susceptible to HIV-1 infection. Likely mechanisms for the superior potency of the CD4-10-17b CAR over the CD4-35-17b CAR include the greater potential of the former to engage in the serial antigen binding required for efficient T cell activation and the ability of two CD4-10-17b molecules to simultaneously bind a single gp120 subunit.


HIV research has been energized by prospects for a cure for HIV infection or, at least, for a "functional cure" whereby antiretroviral therapy can be discontinued without virus rebound. This report describes a novel CD4-based "chimeric antigen receptor" (CAR) which, when genetically engineered into T cells, gives them the capability to selectively respond to and kill HIV-infected cells. This CAR displays enhanced features compared to previously described CD4-based CARs, namely, increased potency and avoidance of the undesired rendering of the genetically modified CD8 T cells susceptible to HIV infection. When adoptively transferred back to the individual, the genetically modified T cells will hopefully provide durable killing of infected cells and sustained virus suppression without continued antiretroviral therapy, i.e., a functional cure.

Jawahar Raina
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A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions.

A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions.


Lentiviruses such as HIV-1 encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs) that include motifs mediating interactions with host-cell-trafficking factors. We demonstrated recently that Rab11-family interacting protein 1C (FIP1C) is required for CT-dependent incorporation of Env into HIV-1 particles. Here, we used viruses bearing targeted substitutions within CT to map the FIP1C-dependent incorporation of Env. We identified YW795 as a critical motif mediating cell-type-dependent Env incorporation. Disruption of YW795 reproduced the cell-type-dependent particle incorporation of Env that had previously been observed with large truncations of CT. A revertant virus bearing a single amino acid change near the C terminus of CT restored wild-type levels of Env incorporation, Gag-Env colocalization on the plasma membrane, and viral replication. These findings highlight the importance of YW795 in the cell-type-dependent incorporation of Env and support a model of HIV assembly in which FIP1C/RCP mediates Env trafficking to the particle assembly site.


FIP1C; HIV assembly; HIV envelope; Rab coupling protein; pseudotyping

Jawahar Raina
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ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.


Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.


endoplasmic reticulum stress (ER stress); endoplasmic-reticulum-associated protein degradation (ERAD); glycoprotein; glycoprotein biosynthesis; human immunodeficiency virus (HIV); protein degradation; unfolded protein response (UPR); viral protein

Jawahar Raina
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Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte-derived macrophages: the importance of HIV-1 envelope V1V2 region.

Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte-derived macrophages: the importance of HIV-1 envelope V1V2 region.


Monocytes and monocyte-derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte-derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor-derived monocyte-derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor-derived monocyte-derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1 interaction with sialic acid-binding immunoglobulin-like lectin and entry into macrophage colony-stimulating factor-derived monocyte-derived macrophages. Removal of sialic acid residues or glycans from scaffolded V1V2 protein decreased human immunodeficiency virus type 1 infectivity. These results highlight the importance of sialic acids on the V1V2 region in binding to sialic acid-binding immunoglobulin-like lectin and suggest that the unusually long surface-exposed sialic acid-binding immunoglobulin-like lectin might aid in the capture and entry of human immunodeficiency virus type 1 into monocyte-derived macrophages.


GM-CSF; M-CSF; flow cytometry; qRT-PCR; surface plasmon resonance; viral entry

Jawahar Raina
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Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.


During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.


Annexin A2; Annexin A2 heterotetramer; HIV-1; Inhibitor; Macrophage; Receptor

Jawahar Raina
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Structural insights into coronavirus entry.

Structural insights into coronavirus entry.


Coronaviruses (CoVs) have caused outbreaks of deadly pneumonia in humans since the beginning of the 21st century. The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and was responsible for an epidemic that spread to five continents with a fatality rate of 10% before being contained in 2003 (with additional cases reported in 2004). The Middle-East respiratory syndrome coronavirus (MERS-CoV) emerged in the Arabian Peninsula in 2012 and has caused recurrent outbreaks in humans with a fatality rate of 35%. SARS-CoV and MERS-CoV are zoonotic viruses that crossed the species barrier using bats/palm civets and dromedary camels, respectively. No specific treatments or vaccines have been approved against any of the six human coronaviruses, highlighting the need to investigate the principles governing viral entry and cross-species transmission as well as to prepare for zoonotic outbreaks which are likely to occur due to the large reservoir of CoVs found in mammals and birds. Here, we review our understanding of the infection mechanism used by coronaviruses derived from recent structural and biochemical studies.


Coronavirus; Fusion protein; Membrane fusion; Proteolytic activation; Spike glycoprotein; Vaccine design

Jawahar Raina
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