Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC 2009 April 1.
Published in final edited form as:
PMCID: PMC2572723

HIV-1 clade-specific differences in the induction of neuropathogenesis


HIV associated dementia (HAD) is common among clade-B HIV-infected individuals, but less common and less severe among individuals infected with clade-C HIV-1, suggesting clade-specific differences in neuropathogenicity. Though differences in neuro-pathogenecity have been investigated in-vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using SCID mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1ADA) or clade C (HIVIndie-C1) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade-C made fewer memory errors than those exposed to HIV-1 clade-B. Histopathological analysis of mice exposed to clade-B exhibited greater astrogliosis and increased loss of neuronal network integrity. In-vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1Indie-C1-infected macrophages recruited monocytes poorly in-vitro compared to that of HIV-1ADA–infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.

Keywords: Dementia, Human, Virus, Cognitive, Lentiviruses, Macrophage


HIV associated dementia (HAD) affected 15 to 30% of infected individuals prior to the widespread use of HAART (highly active anti-retroviral therapy) in the US (McArthur et al., 1993). Despite reduced HAD incidence with the advent of HAART (Sacktor et al., 2001; Dore et al., 2003), improved survival rates of HIV-infected individuals have lead to increase in cognitive impairment due to chronic central nervous system HIV exposure (Sacktor et al., 2001; Langford et al., 2003). In contrast, incidence/prevalence of severe forms of HAD is reported to be considerably lower (2 to 4 percent) in the HAART-naïve HIV-1 infected population in India although milder forms of disease have been reported (Satishchandra et al., 2000; Riedel et al., 2006; Gupta et al., 2007). Considering that the Indian AIDS epidemic is dominated by clade-C HIV-1 (Chakrabarti et al., 2000; Siddappa et al., 2004) and that the clade-C HIV-1 is found in brain autopsies (Mahadevan et al., 2007) from HIV infected Indian patients, suggest a clade-specific difference in the neuropathogenesis of HIV-1. Clade-C HIV-1 is responsible for more than half of new HIV-1 infections worldwide (Geretti, 2006) and a major proportion of HIV infections in India (Siddappa et al., 2004) whereas in North America, HIV-1 clade-B is responsible for nearly all HIV-1 infections.

Mechanisms underlying the pathogenesis of HAD, characterized by neuronal injury, are complex including infiltration of monocyte-derived macrophages (MDM) into brain and neurotoxic effects of HIV-1 proteins (e.g., gp120 and Tat). Tat plays multiple roles in HAD pathogenesis including direct neurotoxicity (Gourdou et al., 1990; Sabatier et al., 1991), chemokine dysregulation (Conant et al., 1998; Weiss et al., 1999) and cytokine induction. A dicysteine motif in Tat has been implicated in direct monocyte chemotaxis in-vitro (Albini et al., 1998). We previously reported that clade-C Tat is divergent from other HIV-1 clades in that the dicysteine motif (C30C31), which is highly conserved in all clades examined, exhibits a C31S polymorphism (Ranga et al., 2004) rendering it defective for monocyte chemotaxis in-vitro.

Differential distribution of B and C HIV-1 clades, differences in host genetics and access to anti-retrovirals make it difficult to assess neuropathologic differences between populations infected by B and C HIV-1 clades. These difficulties are largely overcome by the use of the severe combined immune deficiency mouse HIV encephalitis (SCID HIVE) model (Tyor et al., 1993; Persidsky et al., 1996), in which for the first time the two HIV-1 clades are compared in a single host. This model involves intracranial injection of MDM infected by HIV-1 into SCID mice and its strength lies in its ability to recapitulate key pathological and behavioral features of human HAD (Avgeropoulos et al., 1998; Cook et al., 2005). Importantly, these mice also exhibit behavioral deficits when tested in a water radial arm maze (WRAM) (Avgeropoulos et al., 1998; Sas et al., 2007), which are consistent with cognitive dysfunction in human HAD. In this report, we primarily investigate neuropathogenic differences of clade-B and clade-C HIV-1 isolates. We find that neuropathogenesis by a clade-C HIV-1 results in a milder cognitive dysfunction than with a clade-B HIV-1. We also show that: (i) HIV-infected MDM or the media from such cells can recruit monocytes; (ii) that monocyte recruitment is Tat- and CCL2-dependent; and (iii) that clade-C HIV-1 is significantly compromised for monocyte chemotaxis. Our work shows that both direct and indirect effects of HIV-1 Tat protein are compromised in subtype C HIV-1 in a virological setting.


HIV-1 Infection, Animals and Behavioral Testing

Primary human MDM (purchased from the U. Nebraska Medical Center) were cultured in DMEM (GIBCO) with 10% Human Serum (Sigma Hyclone), Penicillin-Streptomycin (GIBCO) and monocyte-colony stimulating factor (MCSF; Sigma) at 6.6ng/ml at 37°C with 5% CO2 for 7 days in Teflon-coated flasks to prevent attachment. To ensure that the HIV-infected MDMs utilized for intracranial injection had equivalent viral loads, approximately 5 x 106 MDMs were infected with HIV-1ADA (clade-B) at a Multiplicity of Infection (MOI) of 0.1 (1 h) or HIV-1IndieC1 (clade-C) at an MOI of 0.01 (3 h). HIV-1ADA was obtained from the U. Nebraska Medical center and HIV-1IndieC1 prepared by transfecting 293T cells with p93IN101 DNA (Mochizuki et al., 1999). Uninfected MDM were used as controls. Following infection, MDM were resuspended in medium devoid of MCSF and cultured for 14 days with media changes every third day. At the end of 14-day incubation, both sets of MDM displayed similar viral load as determined by immunocytochemistry with anti-p24 antibodies (DAKO) and ELISA to measure p24 levels in the media (Perkin-Elmer) (Figure 2). MDM, grown in Teflon-coated flasks, are recovered by centrifugation and resuspended in PBS for inoculation into mice.

Figure 2
In-vitro and in-vivo quantitation of viral infectivity in injected MDMs and infected mouse brains. a. HIV-1 p24 immunostaining of clade-B and clade-C HIV-infected MDMs in culture prior to injecting into mouse brain. The p24-positive cells are stained ...

Eighteen four week-old C57BL SCID mice (Jackson Laboratory) were acclimatized to the animal room for a week prior to injection. Mice were single-housed in micro-isolator cages (biosafety level-3 equivalent). The animal room was set on a 12 hour light cycle. Cages, bedding, food, and water were sterilized before use. Animal protocols were approved by the Medical University of South Carolina’s Institutional Animal Care and Use Committee. Approximately 1 x 105 HIV-1 infected MDMs (Clade-B or C) or uninfected MDMs were injected into the right frontal lobe of 5-week old SCID mice (n = 6 per group). Six days after intracranial (IC) injections, WRAM behavior testing ensued. This win-shift WRAM utilizes water escape onto hidden platforms as the reinforcer (Hyde et al., 1998; Bimonte-Nelson et al., 2003; Hunter et al., 2004). The 8-arm maze had submerged escape platforms placed on the ends of 4 arms. Each subject had different platform locations that remained fixed throughout the experiment. A subject was released from the start arm and had 2 min to locate a platform. Once a platform was found, the mouse remained on it for 15s, and was then returned to its heated cage for 30s until the next trial. During the interval, the platform found by the mouse was removed from the maze. The animal was then placed again into the start alley and allowed to locate a second platform. The same sequence of events was repeated daily until all four platforms were located. Thus, for each animal, a daily session consisted of four trials, with the number of platformed arms reduced by one on each subsequent trial resulting in the working memory system becoming increasingly demanding as trials progressed. Animals received 4 trials per day for 12 days. The testing protocol included an initial learning phase (days 1–8) and a final testing phase (days 9–12). Working memory correct (WMC) and working memory incorrect (WMI) errors were quantified based on Jarrard et al.’s orthogonal measures (Jarrard, 1993; Hyde et al., 1998; Bimonte-Nelson et al., 2003; Hunter et al., 2004). WMC errors were defined as entries into an arm wherein a platform had already been located and WMI errors were defined as repeat entries into an arm that never contained a platform.

Immunohistochemistry, densitometry and detection of HIV RNA levels using Real-time PCR

Following WRAM testing, mice were sacrificed and brains extracted and frozen for immunohistochemistry (n = 6). Approximately 13 sets of serial five micron, coronal sections were taken from the frontal through the temporo-parietal lobes of each mouse. Each set was separated by 46 intervening 5 micron sections, which were saved for RNA extraction. Each stained set consisted of slides that underwent immunoperoxidase staining separately for human macrophages (EBM-11, Dako); HIV-1 p24 (p24, DAKO); astrocytes (GFAP, Chemicon) and neuronal dendrites (MAP2, Chemicon) as described (Avgeropoulos et al., 1998; Sas et al., 2007). Average number of primary human macrophages (EBM-11 positive cells) and HIV-infected cells (p24 positive cells) were determined by light microscopy. Immunoperoxidase stained slides were imaged at 20x using an Olympus microscope. Densitometry analysis was performed to determine the intensity of immunoperoxidase stain using NIH Image software on the images (20x) representing sections stained for astrogliosis (GFAP) and neuronal network integrity (MAP-2) (Sas et al., 2007). For measuring neuronal integrity (MAP-2), the left hemisphere (non-injected hemisphere) of each mouse served as a normal control and was set at 100%. The measured value of the right hemisphere was compared to the left hemisphere of the section to determine the relative differences in MAP2 staining. Three sections per mouse from 6 mice per group were analyzed in the above manner to derive the final values plotted in Figure 3.

Figure 3
Quantitative assessment of immunoperoxidase stained sections of HIV-infected and control mouse brains a. Measurement of GFAP-positive cells to determine astrogliosis. Densitometric analysis of five frontal cortex GFAP stained sections from the brains ...

For real time PCR measurements of viral RNA, the tissue from these intervening sections was homogenized and the RNA was extracted according to the RNAwiz (Ambion, Austin, TX) protocol. Approximately 1μg total RNA was used for complementary DNA (cDNA) synthesis. First-strand cDNA synthesis was performed using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). HIV-1 gag RNA levels were determined using real-time polymerase chain reaction with ABI 7000 prism (Applied Biosystems) to determine viral load, according to manufacturer’s suggested protocol.

Measurement of monocyte migration using a dual chamber assay

Human MDM were cultured for 4 days under the same conditions as described above. About 2 x 105 of MDM were plated in the lower wells of a 24-well plate. (Rao et al., 2009) Approximately 2 x 105 cultured MDM were infected with HIV-1ADA virus (clade-B) at a MOI of 0.1 for 1 h or HIV-1IndieC1 (clade-C) at an MOI of 0.01 for 3 hours and incubated for 5 days in the above-mentioned medium devoid of MCSF. ELISA for p24 in the supernatant staining and p24 staining of macrophages were used to ensure equal viral load for both HIV-1ADA and HIV-1 IndieC1. In the upper chamber, 2 X 105 elutriated human monocytes were added. To facilitate their identification subsequent to migration, monocytes were stained with CFSE dye prior to adding them to the transwells. After 24 h incubation, cells in the lower chamber were examined in a fluorescence microscope to quantify the migrated cells.

Neutralization of Tat and CCL2 proteins

Neutralization of Tat and CCL2 proteins was carried out by a slightly modified version of the earlier technique (Weiss et al., 1999). Four days post HIV-1 infection, the infected MDM prepared the same way as described in the migration experiments, were incubated with fresh medium (DMEM 2% Human serum) for 24 h. (Rao et al., 2009) The conditioned medium (supernatant from HIV-1 infected and Control wells) was then incubated for 1 h with Pansorbin beads (Calbiochem) to which either anti-Tat (E1.1, which neutralizes both Tat proteins - U. Ranga, unpublished observations) or anti-CCL2 antibodies (MAb 279; R & D Systems) were pre-bound. Optimal antibody concentrations were determined for Tat by testing increasing concentrations of antibodies and for CCL2 based on the CCL2 levels in the medium determined by ELISA. Beads were removed by pelleting and the supernatant used in migration experiments following the above mentioned migration protocol.

Tat-induced and HIV-infection induced secretion of CCL2 from MDM and astrocytes

Four days after HIV-infection, MDMs were incubated for 24 hours in fresh medium without human serum to eliminate the contribution of CCL2 in human serum. The Day-5 no serum supernatant was collected and ELISA for CCL2 (R&D Systems) was performed in triplicates in 3 separate experiments as described before (Eugenin et al., 2006).

Statistical analysis

Statistical analysis for the behavior study was via StatView. Repeated measures ANOVA analysis was performed and the p values obtained by comparing errors made by clade-B, clade-C and control mice individually to each other. For migration studies, chemokine expression and histopathology analysis, the significance and p values were determined using Microsoft Excel.


SCID mice with clade-C HIV-1 display milder cognitive deficits than clade-B HIV SCID mice

The SCID mouse HIV encephalitis model, developed originally by Tyor et al. (Tyor et al., 1993) and subsequently improved by both W. Tyor and coworkers and H. Gendelman and coworkers, is a useful, sensitive and well characterized animal models to test HIV-induced neurotoxicity and behavioral changes. (Persidsky et al., 1996; Persidsky et al., 1997; Avgeropoulos et al., 1998; Cook et al., 2005; Sas et al., 2007). Behavioral testing with WRAM revealed that clade-B HIVE mice exhibited poor working memory as shown on two orthogonal measures of working memory competence. Clade-B HIVE mice made more working memory incorrect (WMI; a form of long-term memory) and working memory correct (WMC; a form of short-term memory) errors when compared to controls (Figure 1). Repeated measures ANOVA to analyze the number of errors, which were collapsed across testing days (days 9–12) and trials revealed that clade-B HIVE mice made significantly more WMI errors when compared to clade-C HIVE mice (p = 0.046; Figure 1a) or the controls (p = 0.005; Figure 1a) injected with uninfected MDM. Clade-B HIVE mice made significantly more WMC errors as compared to the controls (p = 0.042; Figure 1b). Clade-C HIVE mice made an intermediate number of WMI errors between clade-B and the controls (Figure 1b). Moreover, clade-B HIVE mice were less successful in handling an increasing working memory load as shown on two dependent variables. Indeed clade-B HIVE mice made significantly more WMC and WMI errors than clade-C HIVE or the control mice as the memory load increased during the later trials (Figures 1c, 1d).

Figure 1
Cognitive performance of HIV-infected and control SCID mice on the latter phase of testing. a, b. Cognitive performance of HIV-infected and control mice averaged across days (9 to 12) and trials, 1-4 trials for WMI (a form of long-term memory; Panel a) ...

Brains of SCID mice exposed to either HIV-1 clade-B or C show similar viral loads, but those with clade-C HIV-1 show decreased pathology

Evaluating different clades of HIV-1 for their potential to induce SCID HIVE requires that the viral load of these related, but distinct, viruses are comparable. Therefore, in addition to ensuring that the MDM injected into mouse brain contained equal proportions of HIV-positive cells with similar viral replication (Figure 2), immunohistochemical analysis of the frozen sections was performed with antibodies to HIV-1 p24 (Figure 2). This analysis showed equal numbers of HIV-positive cells in the brains of SCID mice infected with either virus (11 ± 4 vs. 13±5 (mean ± S.D) p24 positive cells/section for clade-B and clade-C HIV-infected SCID mouse brains respectively; the data represent an average of 5 sections per mouse brain around the injection site in the frontal cortex region and 9 mice in each group). RT-PCR analysis of HIV-1 clade-B and clade-C infected mouse brains (n = 6; from fontal cortex) revealed average log copy numbers of 12. 75 ± 0.11 and 12.36 ± 0.16 (mean ± S.E.M) respectively (Figure 2).

Astrogliosis, as evidenced by GFAP staining, and a decrease in neuronal network integrity, as indicated by reduction in MAP2-staining, were observed in the brains of mice injected with HIV-infected MDM in frontal lobe sections around the injection tract. Brains of control mice also displayed low levels of astrogliosis and a slight reduction in MAP-2 staining suggesting background inflammation. However, the severity of astrogliosis in the frontal cortex region was greatest in clade-B HIV-infected SCID mice (Figure 3a) when compared to clade-C HIVE (p = 0.0117) and control (p = 0.0008) mice. Percentage MAP2 staining comparing the right and the left frontal cortex regions for neuronal integrity revealed a significant difference between brains of mice injected with clade-B HIV-1 and control brains (p value = 0.0006) but no statistically significant differences between the two HIV-1 clades (Figure 3b). Nevertheless, a trend for less severe MAP2 changes was observed in clade-C HIVE mice compared to clade-B HIV-1 HIVE mice.

Clade-C HIV-infected MDMs display reduced monocyte chemotaxis

Monocyte infiltration into the brain is a hallmark of HAD. Although Tat causes monocyte chemotaxis in-vitro, monocyte chemotaxis by Tat in the context of HIV-infected cells has not been demonstrated. To investigate clade differences in monocyte chemotaxis, we employed MDM, HIV-1 susceptible cells that are relevant to HIV-1 neuropathogenesis, that were infected with either HIV-1ADA (clade-B) or HIV-1IndieC1 (clade-C). HIV-infected MDM were added to the bottom chamber of the trans-well system and elutriation-derived, undifferentiated monocytes to the top chamber. (Rao et al., 2009) Following 24h incubation, we observed that clade-B infected MDMs attracted 64% (p = 3.5X10−5) more monocytes than did clade-C infected MDM (Figure 4a). Supernatant p24 ELISA (Perkin-Elmer) measurements were done prior to the migration experiments to ensure that the two sets of MDM cells had similar viral loads.

Figure 4
Monocyte migration assays in the presence of HIV-1 infected MDMs using a dual-chamber assay. a. Monocyte migration caused by clade-B and clade-C HIV-infected MDM. The numbers plotted reflect the monocytes detected in the lower chamber by counting the ...

Chemotaxis induced by HIV-infected MDMs is mediated by Tat and CCL2

HIV-infected MDM secrete a number of soluble factors into the medium including Tat protein. To delineate the contribution of factors influencing monocyte chemotaxis, the medium from infected MDM cell culture was first confirmed to induce chemotaxis using the in vitro dual chamber assay described earlier (Figure 4b; no treatment). Then, to determine if extracellular Tat plays a role in chemotaxis, the medium was immunodepleted using anti-Tat neutralizing antibodies. These experiments demonstrated that a greater proportion of monocytes recruited by clade-B HIV-infected MDM were due to Tat when compared to clade-C HIV-infected MDM. Similarly, immunodepletion of CCL2 revealed that CCL2 also plays a key role in monocyte chemotaxis due to infection by either clade of HIV-1 (Figure 4b).

Poor induction of CCL2 chemokine by clade-C HIV-1 and its Tat protein

Tat released from infected cells can gain access into uninfected cells in the vicinity (Schwarze et al., 1999). A transcriptional transactivator of both viral and cellular genes, Tat induces astrocytes and monocytes in vitro to produce chemokines (e.g. CCL2) (Conant et al., 1998; Weiss et al., 1999; D’Aversa et al., 2004). Therefore, we investigated the mechanistic basis for lowered monocyte chemotaxis by clade-C Tat protein. We measured the production of CCL2 by monocytes and astrocytes following the addition of Tat protein from either clade to cultures for 24 hours. Clade-C Tat protein, as compared to clade-B Tat, induced significantly lower levels of CCL2, in a dose-dependent manner, in both MDM (100ng Tat, p = 1.4x10−5; 500ng Tat, p = 6.1x10−10) and astrocytes (100ng Tat, p = 0.0017; 500ng Tat, p = 0.0001) (Figure 5a, 5b).

Figure 5
Differential Induction of CCL2 by HIV infected and Tat treated MDMs and astrocytes (in-vitro): a. Measurement of CCL2 secreted by MDM exposed to Tat protein from clade-B or clade-C HIV-1 at 100ng/ml and 500ng/ml. Control represents cells incubated with ...

To test the relevance of this finding in the context of HIV-1 replication, we measured the secretion of CCL2 by clade-B and C HIV-1-infected MDM. HIV-infected MDM were incubated for 24h in serum-free medium to eliminate the confounding presence of CCL2 in human serum employed for growing MDM. Clade-C HIV-infected MDM displayed a significantly reduced ability to secrete CCL2 in comparison with clade-B-HIV-infected MDM (p = 0.0124) (Figure 5c).


Reports of reduced prevalence of severe forms HAD in regions with predominantly clade-C HIV-infections, such as India (Satishchandra et al., 2000; Riedel et al., 2006; Gupta et al., 2007), imply clade-specific differences in neuropathogenesis. To verify this hypothesis, we have employed in-vitro experiments with purified Tat proteins and in-vivo experiments using infectious molecular clones of HIV-1 in the SCID HIVE model. We have extended our previous findings that clade-C Tat is defective in its chemotactic properties. Our results demonstrate, for the first time, differences in neuropathogenesis between clade-B and C HIV-1 isolates in an in-vivo model. Differential neuropathogenesis observed for the two clades in-vivo also correlated with differential ability of HIV-infected MDM to recruit monocytes in-vitro and in inducing chemokines such as CCL2 in infected cells.

Our results revealed significant differences between the two clades in terms of behavioral deficits. Clade-B HIVE mice made most working me mory errors and clade-C HIVE mice made an intermediate number of errors in reference to clade B and control mice. Our approach of injecting HIV-infected macrophages into the frontal cortex produces well-documented neuropathological and behavioral effects that are widespread rather than limited to the site of injection or right side alone. As previously described (Griffin et al., 2004), multiple areas of the brain are likely involved in causing the behavioral abnormalities, including frontal lobes, hippocampus, and basal ganglia. Some of these affects require a relatively remote action of HIV. These effects are thought to be related to diffusible substances produced by HIV infected macrophages and activated glia (Griffin et al., 2004). Our behavioral testing protocol does not include WRAM testing of uninoculated normal SCID mice as they have been previously shown (Avgeropoulos et al., 1998) to perform consistently with, or even better than, our controls injected with uninfected MDMs.

MAP2 staining serves both as a means of measuring HIV induced damage to neuronal network integrity (Avgeropoulos et al., 1998; Zheng et al., 2001; Dou et al., 2003; O’Donnell et al., 2006) as well as a histopathological correlate of HIV induced behavioral abnormalities. (Cook-Easterwood et al., 2007; Sas et al., 2007) Results from analysis of MAP-2 stained frontal cortex regions parallel the behavioral findings. Loss of MAP-2 staining produced by clade-C HIV-1 is intermediate compared to clade-B and control mice. Astrogliosis, a key pathological feature of HAD, was significantly greater (p = 0.0117) in clade-B HIVE mice compared to clade-C HIVE mice. Similar number of p24-positive cells in the brains of both types of mice confirmed equal viral loads in both cases. These findings not only suggest that clade differences play a major role in pathogenesis of HAD, but also highlight the usefulness of the SCID HIVE mouse model in detecting subtle differences in behavior and histopathology.

More recently it has been shown that direct neurotoxicity of Tat may be mediated by Tat’s binding to NMDA receptor subunits NR1 and 2A (Avindra Nath, personal communication) and that clade-C or a C31S mutant of clade-B Tat protein does not cause neurotoxicity (Li et al., 2005), providing an additional explanation for clade differences in NeuroAIDS. The mechanism of toxicity was not mediated by a complex formation among LRP, PSD-95, NMDAR and nNOS on the surface of human neurons, as we described previously (Eugenin et al., 2007). It was also not due to LRP-mediated Tat-internalization as described in neurons (Liu et al., 2000), because RAP, an LRP blocker, did not abrogate toxicity. These differences in Tat-mediated toxicity may be due to the low expression of LRP receptor on HEK cells and the absence of PSD-95 to bridge NMDA receptors and LRP.

Irrespective of the precise mechanism of Tat-mediated effects on neurons, our results show that behavioral and neuropathological differences between HIV-1 clades are due to an attenuated ability of clade-C Tat to cause neurotoxicity by both direct and indirect mechanisms. While the studies by A. Nath and colleagues (personal communication) point to clade-differences in NMDA receptor-mediated apoptosis, Campbell et al. showed that clade-C Tat protein is defective in its ability to generate an intracellular calcium influx and in inducing TNF-α(Campbell et al., 2007), a cytokine which promotes astrogliosis. Additionally, Mishra et al. have reported lower neurotoxicity, a significantly reduced reactive oxygen species (ROS) induction and weaker mitochondrial membrane polarization in human neurons by clade-C Tat (Mishra, 2007).

These neurotoxic effects of clade-B HIV-1 may have implications for both neuronal and astrocytic dysfunction in HIVE in SCID mice and HAD in humans. The increased astrogliosis seen in HIVE mice with clade-B HIV-1 compared to C reflect a larger pool of dysfunctional astrocytes (Lipton and Gendelman, 1995) observed. Normally, astrocytes are responsible for clearing excess glutamate at the synaptic cleft to prevent NMDA receptor-mediated excitotoxicity. However, if astrocytes are impaired in this function, it could explain why neurons in the area of greatest astrogliosis display decreased dendritic arborization. In addition, neurotoxicity in clade B mice could have more direct affects on neurons that are in the vicinity of HIV-infected human MDM, adding to (indirect) astrocyte affects on neuronal dendritic formation. Therefore, since frontal cortical dendritic formation is important to learned behavior, the clade-B mice make a greater number of errors in the WRAM.

Previously, we showed that clade-C Tat is defective in monocyte chemotaxis using purified Tat protein in-vitro (Ranga et al., 2004) Here, we utilized HIV-1 infected MDM for the recruitment of monocytes to demonstrate that monocyte chemotaxis occurs in the setting of virus-infected cells. In comparing the two clades of HIV-1, the differences observed were not caused by differential virus growth. Under these conditions, clade-C HIV-infected cells were able to recruit significantly reduced numbers of monocytes compared to clade-B HIV-infected MDM. The SCID HIVE model, employed here does not allow us to examine whether the differential monocyte chemotaxis observed in vitro applies to monocyte migration across the blood brain barrier in vivo. Therefore we are looking into developing of modified mouse models, in which, one can observe the migration of macrophages introduced in the periphery to the sites in brain where HIV-infected macrophages reside under conditions that transiently break down the blood-brain barrier.

In verifying that the monocyte chemotaxis induced by HIV-infected MDMs is truly mediated by Tat protein, we employed anti-Tat antibodies with a proven ability to neutralize both clade-B and clade-C Tat proteins (Siddappa et al., 2006). Interestingly, neutralizing the Tat protein present in the spent media lowered monocyte migration to control levels and neutralizing the CCL2 was also effective in blocking monocyte migration – suggesting that Tat or HIV-infection induces the secretion of CCL2 which then mediates monocyte migration. These novel findings demonstrate a direct role for Tat during HIV-infection of MDM in monocyte migration. CCL2 is presumably produced as a result of HIV-1 infection. Media from clade-C HIV-infected MDM also showed reduction in the migrating monocytes with anti-Tat and anti-CCL2 antibodies. This reduction was lower than that observed with clade-B, but statistically significant (p = 008 for anti-Tat and p = 0.005 for anti-CCL2).

The ability of clade-B HIV-1 Tat protein as well as clade-B HIV-1 virus to induce MDM and astrocytes to secrete CCL2 points to an indirect means by which HIV-infection leads to enhanced monocyte infiltration into the brain. We took advantage of the availability of Tat preparations that were equally biologically active in their ability to be taken up by cells and to activate HIV-1 LTR-mediated expression of a GFP reporter (Siddappa et al., 2006). Despite their similar activities (Siddappa et al., 2006), there were significant differences in CCL2 induction by the two different Tat proteins when incubated with primary astrocytes or MDM. These differences were also observed between MDM infected with the two clades of HIV-1. The differential induction of CCL2 by the two Tat proteins and by infection with the two clades of HIV-1 suggests another key difference between the clades in the pathogenesis of HAD.

Our results demonstrate that genotypic differences between HIV-1 clade-B and clade-C viruses do translate to phenotype differences in HAD pathogenesis outcomes in-vivo by virtue of decreased direct neurotoxicity of clade-C HIV-1 as well as lead to differences observable in-vitro, including defective monocyte migration and the impaired secretion of chemotactic proteins such as CCL2. Our results provides a concrete proof of inter-clade differences and hope that specific HIV genes directly involved in neuropathogenesis can be pinpointed by the use of chimeric viruses between clades, work on which is currently being pursued in our lab. The findings reported here may have significant implications for future treatment strategies in HAD by helping target specific genes responsible for pathogenesis of HAD.


The authors wish to thank the Einstein/MMC Center for AIDS Research (CFAR) for the use of Immunology/Pathology and BSL3/Clinical Virology core services, Albert Einstein Cancer Center for the use of histopathology core and Adam Davis for reading the manuscript. The research described in this report was supported by Public Health Service research grants to V.R.P. (R21 MH075636, RO1 MH083579). A.S. is supported by an NIH pre-doctoral fellowship (F31 NS054592) and E.A.E. by a mentoring grant (KO1 MH076679). Housing of mice at MUSC was partially supported by NIH C06 RR015455.



There are no competing interests


  • Albini A, Benelli R, Giunciuglio D, Cai T, Mariani G, Ferrini S, Noonan DM. Identification of a novel domain of HIV tat involved in monocyte chemotaxis. J Biol Chem. 1998;273:15895–15900. [PubMed]
  • Avgeropoulos N, Kelley B, Middaugh L, Arrigo S, Persidsky Y, Gendelman HE, Tyor WR. SCID mice with HIV encephalitis develop behavioral abnormalities. J Acquir Immune Defic Syndr Hum Retrovirol. 1998;18:13–20. [PubMed]
  • Bimonte-Nelson HA, Hunter CL, Nelson ME, Granholm AC. Frontal cortex BDNF levels correlate with working memory in an animal model of Down syndrome. Behav Brain Res. 2003;139:47–57. [PubMed]
  • Campbell GR, Watkins JD, Singh KK, Loret EP, Spector SA. Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes. J Virol. 2007;81:5919–5928. [PMC free article] [PubMed]
  • Chakrabarti S, Panda S, Chatterjee A, Sarkar S, Manna B, Singh NB, Naik TN, Detels R, Bhattacharya SK. HIV-1 subtypes in injecting drug users & their non-injecting wives in Manipur, India. Indian J Med Res. 2000;111:189–194. [PubMed]
  • Conant K, Garzino-Demo A, Nath A, McArthur JC, Halliday W, Power C, Gallo RC, Major EO. Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia. Proc Natl Acad Sci U S A. 1998;95:3117–3121. [PubMed]
  • Cook JE, Dasgupta S, Middaugh LD, Terry EC, Gorry PR, Wesselingh SL, Tyor WR. Highly active antiretroviral therapy and human immunodeficiency virus encephalitis. Ann Neurol. 2005;57:795–803. [PubMed]
  • Cook-Easterwood J, Middaugh LD, Griffin WC, 3rd, Khan I, Tyor WR. Highly active antiretroviral therapy of cognitive dysfunction and neuronal abnormalities in SCID mice with HIV encephalitis. Exp Neurol. 2007;205:506–512. [PMC free article] [PubMed]
  • D’Aversa TG, Yu KO, Berman JW. Expression of chemokines by human fetal microglia after treatment with the human immunodeficiency virus type 1 protein Tat. J Neurovirol. 2004;10:86–97. [PubMed]
  • Dore GJ, McDonald A, Li Y, Kaldor JM, Brew BJ. Marked improvement in survival following AIDS dementia complex in the era of highly active antiretroviral therapy. Aids. 2003;17:1539–1545. [PubMed]
  • Dou H, Birusingh K, Faraci J, Gorantla S, Poluektova LY, Maggirwar SB, Dewhurst S, Gelbard HA, Gendelman HE. Neuroprotective activities of sodium valproate in a murine model of human immunodeficiency virus-1 encephalitis. J Neurosci. 2003;23:9162–9170. [PubMed]
  • Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, Berman JW. CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. J Neurosci. 2006;26:1098–1106. [PubMed]
  • Eugenin EA, King JE, Nath A, Calderon TM, Zukin RS, Bennett MV, Berman JW. HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes. Proc Natl Acad Sci U S A. 2007;104:3438–3443. [PubMed]
  • Geretti AM. HIV-1 subtypes: epidemiology and significance for HIV management. Curr Opin Infect Dis. 2006;19:1–7. [PubMed]
  • Gourdou I, Mabrouk K, Harkiss G, Marchot P, Watt N, Hery F, Vigne R. Neurotoxicity in mice due to cysteine-rich parts of visna virus and HIV-1 Tat proteins. C R Acad Sci III. 1990;311:149–155. [PubMed]
  • Griffin WC, 3rd, Middaugh LD, Cook JE, Tyor WR. The severe combined immunodeficient (SCID) mouse model of human immunodeficiency virus encephalitis: deficits in cognitive function. J Neurovirol. 2004;10:109–115. [PubMed]
  • Gupta JD, Satishchandra P, Gopukumar K, Wilkie F, Waldrop-Valverde D, Ellis R, Ownby R, Subbakrishna DK, Desai A, Kamat A, Ravi V, Rao BS, Satish KS, Kumar M. Neuropsychological deficits in human immunodeficiency virus type 1 clade C-seropositive adults from South India. J Neurovirol. 2007;13:195–202. [PubMed]
  • Hunter CL, Bimonte-Nelson HA, Nelson M, Eckman CB, Granholm AC. Behavioral and neurobiological markers of Alzheimer’s disease in Ts65Dn mice: effects of estrogen. Neurobiol Aging. 2004;25:873–884. [PubMed]
  • Hyde LA, Hoplight BJ, Denenberg VH. Water version of the radial-arm maze: learning in three inbred strains of mice. Brain Res. 1998;785:236–244. [PubMed]
  • Jarrard LE. On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol. 1993;60:9–26. [PubMed]
  • Langford TD, Letendre SL, Larrea GJ, Masliah E. Changing patterns in the neuropathogenesis of HIV during the HAART era. Brain Pathol. 2003;13:195–210. [PubMed]
  • Li W, Galey D, Mattson MP, Nath A. Molecular and cellular mechanisms of neuronal cell death in HIV dementia. Neurotox Res. 2005;8:119–134. [PubMed]
  • Lipton SA, Gendelman HE. Dementia associated with the acquired immunodeficiency syndrome. Seminars in medicine of the Beth Israel Hospital, Boston. N Engl J Med. 1995;332:934–940. [PubMed]
  • Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A, He JJ. Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med. 2000;6:1380–1387. [PubMed]
  • Mahadevan A, Shankar SK, Satishchandra P, Ranga U, Chickabasaviah YT, Santosh V, Vasanthapuram R, Pardo CA, Nath A, Zink MC. Characterization of human immunodeficiency virus (HIV)-infected cells in infiltrates associated with CNS opportunistic infections in patients with HIV clade C infection. J Neuropathol Exp Neurol. 2007;66:799–808. [PubMed]
  • McArthur JC, Hoover DR, Bacellar H, Miller EN, Cohen BA, Becker JT, Graham NM, McArthur JH, Selnes OA, Jacobson LP, et al. Dementia in AIDS patients: incidence and risk factors. Multicenter AIDS Cohort Study. Neurology. 1993;43:2245–2252. [PubMed]
  • Mishra M. Clade Specific Neurotoxicity of HIV Tat: Significance of Dicysteine C30C31 Motif. Ann Neurol 2007 [PubMed]
  • Mochizuki N, Otsuka N, Matsuo K, Shiino T, Kojima A, Kurata T, Sakai K, Yamamoto N, Isomura S, Dhole TN, Takebe Y, Matsuda M, Tatsumi M. An infectious DNA clone of HIV type 1 subtype C. AIDS Res Hum Retroviruses. 1999;15:1321–1324. [PubMed]
  • O’Donnell LA, Agrawal A, Jordan-Sciutto KL, Dichter MA, Lynch DR, Kolson DL. Human immunodeficiency virus (HIV)-induced neurotoxicity: roles for the NMDA receptor subtypes. J Neurosci. 2006;26:981–990. [PubMed]
  • Persidsky Y, Buttini M, Limoges J, Bock P, Gendelman HE. An analysis of HIV-1-associated inflammatory products in brain tissue of humans and SCID mice with HIV-1 encephalitis. J Neurovirol. 1997;3:401–416. [PubMed]
  • Persidsky Y, Limoges J, McComb R, Bock P, Baldwin T, Tyor W, Patil A, Nottet HS, Epstein L, Gelbard H, Flanagan E, Reinhard J, Pirruccello SJ, Gendelman HE. Human immunodeficiency virus encephalitis in SCID mice. Am J Pathol. 1996;149:1027–1053. [PubMed]
  • Ranga U, Shankarappa R, Siddappa NB, Ramakrishna L, Nagendran R, Mahalingam M, Mahadevan A, Jayasuryan N, Satishchandra P, Shankar SK, Prasad VR. Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine. J Virol. 2004;78:2586–2590. [PMC free article] [PubMed]
  • Rao VR, Eugenin EA, Berman JW, Prasad VR. Methods to study monocyte migration induced by HIV-infected cells. In: Prasad VR, Kalpana GV, editors. HIV Protocols. Springer; 2009. [PubMed]
  • Riedel D, Ghate M, Nene M, Paranjape R, Mehendale S, Bollinger R, Sacktor N, McArthur J, Nath A. Screening for human immunodeficiency virus (HIV) dementia in an HIV clade C-infected population in India. J Neurovirol. 2006;12:34–38. [PubMed]
  • Sabatier JM, Vives E, Mabrouk K, Benjouad A, Rochat H, Duval A, Hue B, Bahraoui E. Evidence for neurotoxic activity of tat from human immunodeficiency virus type 1. J Virol. 1991;65:961–967. [PMC free article] [PubMed]
  • Sacktor N, Lyles RH, Skolasky R, Kleeberger C, Selnes OA, Miller EN, Becker JT, Cohen B, McArthur JC. HIV-associated neurologic disease incidence changes:: Multicenter AIDS Cohort Study, 1990–1998. Neurology. 2001;56:257–260. [PubMed]
  • Sas A, Nelson HB-N, Tyor WR. Cognitive Dysfunction in HIV Encephalitic SCID mice correlates with levels of Interferon-α in the Brain. AIDS. 2007 In Press. [PubMed]
  • Satishchandra P, Nalini A, Gourie-Devi M, Khanna N, Santosh V, Ravi V, Desai A, Chandramuki A, Jayakumar PN, Shankar SK. Profile of neurologic disorders associated with HIV/AIDS from Bangalore, south India (1989–96) Indian J Med Res. 2000;111:14–23. [PubMed]
  • Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999;285:1569–1572. [PubMed]
  • Siddappa NB, Venkatramanan M, Venkatesh P, Janki MV, Jayasuryan N, Desai A, Ravi V, Ranga U. Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein. Retrovirology. 2006;3:53. [PMC free article] [PubMed]
  • Siddappa NB, Dash PK, Mahadevan A, Jayasuryan N, Hu F, Dice B, Keefe R, Satish KS, Satish B, Sreekanthan K, Chatterjee R, Venu K, Satishchandra P, Ravi V, Shankar SK, Shankarappa R, Ranga U. Identification of subtype C human immunodeficiency virus type 1 by subtype-specific PCR and its use in the characterization of viruses circulating in the southern parts of India. J Clin Microbiol. 2004;42:2742–2751. [PMC free article] [PubMed]
  • Tyor WR, Power C, Gendelman HE, Markham RB. A model of human immunodeficiency virus encephalitis in scid mice. Proc Natl Acad Sci U S A. 1993;90:8658–8662. [PubMed]
  • Weiss JM, Nath A, Major EO, Berman JW. HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes. J Immunol. 1999;163:2953–2959. [PubMed]
  • Zheng J, Thylin MR, Cotter RL, Lopez AL, Ghorpade A, Persidsky Y, Xiong H, Leisman GB, Che MH, Gendelman HE. HIV-1 infected and immune competent mononuclear phagocytes induce quantitative alterations in neuronal dendritic arbor: relevance for HIV-1-associated dementia. Neurotox Res. 2001;3:443–459. [PubMed]