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Clade C HIV-1 isolates circulating in Southern Africa exhibit a greater frequency of dicysteine motif-containing Tat variants than those in Southeast Asia and cause increased neurovirulence.

Rao VR, Neogi U, Talboom JS, Padilla L, Rahman M, Fritz-French C, Gonzalez-Ramirez S, Verma A, Wood C, Ruprecht RM, Ranga U, Azim T, Joska J, Eugenin E, Shet A, Bimonte-Nelson H, Tyor WR, Prasad VR - Retrovirology (2013)

Bottom Line: The proportion of HIV - 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries.In vitro assays revealed that the Southern African isolate, HIV-1C 1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C.Our results indicate that Tat dicysteine motif determines neurovirulence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

ABSTRACT

Background: HIV-1 Clade C (Subtype C; HIV-1C) is responsible for greater than 50% of infections worldwide. Unlike clade B HIV-1 (Subtype B; HIV-1B), which is known to cause HIV associated dementia (HAD) in approximately 15% to 30% of the infected individuals, HIV-1C has been linked with lower prevalence of HAD (0 to 6%) in India and Ethiopia. However, recent studies report a higher prevalence of HAD in South Africa, Zambia and Botswana, where HIV-1C infections predominate. Therefore, we examined whether Southern African HIV-1C is genetically distinct and investigated its neurovirulence. HIV-1 Tat protein is a viral determinant of neurocognitive dysfunction. Therefore, we focused our study on the variations seen in tat gene and its contribution to HIV associated neuropathogenesis.

Results: A phylogenetic analysis of tat sequences of Southern African (South Africa and Zambia) HIV isolates with those from the geographically distant Southeast Asian (India and Bangladesh) isolates revealed that Southern African tat sequences are distinct from Southeast Asian isolates. The proportion of HIV - 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries. Neuropathogenic potential of a Southern African HIV-1C isolate (from Zambia; HIV-1C 1084i), a HIV-1C isolate (HIV-1 IndieC1) from Southeast Asia and a HIV-1B isolate (HIV-1 ADA) from the US were tested using in vitro assays to measure neurovirulence and a SCID mouse HIV encephalitis model to measure cognitive deficits. In vitro assays revealed that the Southern African isolate, HIV-1C 1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C. In neurocognitive tests, SCID mice injected with MDM infected with Southern African HIV-1C 1084i showed greater cognitive dysfunction similar to HIV-1B but much higher than those exposed to Southeast Asian HIV - 1C.

Conclusions: We report here, for the first time, that HIV-1C from Southern African countries is genetically distinct from Southeast Asian HIV-1C and that it exhibits a high frequency of variants with dicysteine motif in a key neurotoxic HIV protein, Tat. Our results indicate that Tat dicysteine motif determines neurovirulence. If confirmed in population studies, it may be possible to predict neurocognitive outcomes of individuals infected with HIV-1C by genotyping Tat.

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Effect of HIV-infected MDM supernatants on neuronal apoptosis. A. HIV-1 infected MDM and uninfected MDM supernatant were diluted with 100 μl of neurobasal medium and incubated with primary human neurons in Matek plates for 18 hours. HIV-1BADA and HIV-1C1084i treatments lead to a greater loss of neuronal viability when compared to HIV-1CIndieC1 and uninfected supernatant. B. Tunnel stained primary human neurons co-stained with anti-neurotubulin antibodies and DAPI (Prolong Gold antifader with DAPI, Invitrogen). The four panels show representative fields for neurons treated with HIV-1BADA, HIV-1C1084i, HIV-1CIndieC1 and media from uninfected macrophages from left to right respectively. The individual (or all) channels are indicated at left. * p < 0.05.
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Figure 4: Effect of HIV-infected MDM supernatants on neuronal apoptosis. A. HIV-1 infected MDM and uninfected MDM supernatant were diluted with 100 μl of neurobasal medium and incubated with primary human neurons in Matek plates for 18 hours. HIV-1BADA and HIV-1C1084i treatments lead to a greater loss of neuronal viability when compared to HIV-1CIndieC1 and uninfected supernatant. B. Tunnel stained primary human neurons co-stained with anti-neurotubulin antibodies and DAPI (Prolong Gold antifader with DAPI, Invitrogen). The four panels show representative fields for neurons treated with HIV-1BADA, HIV-1C1084i, HIV-1CIndieC1 and media from uninfected macrophages from left to right respectively. The individual (or all) channels are indicated at left. * p < 0.05.

Mentions: We next examined the neurotoxicity of HIV-1C1084i Tat when compared to HIV-1IndieC1 TatC and TatB in vitro. Primary human neurons were treated with conditioned medium obtained from HIV-infected MDM and neuronal viability was quantified by using TUNEL assay. Immunofluorescence was used to specifically detect neurons (via staining for neurotubulin), apoptotic cells (TUNEL staining) and nuclei (DAPI staining). The neuronal viability was reduced by 60% when HIV-1C1084i–infected MDM supernatants were used and greater than 80% when HIV-1BADA–infected MDM supernatants were used (Figure 4A and 4B). Media from uninfected Macrophages and HIV-1CIndieC1-infected MDMs resulted in 12% to 25% cell death respectively (Figure 4A, 4B). In order to determine the relative contribution of Tat to neurotoxicity, the MDM supernatants were first depleted of virions and free gp120 using broadly neutralizing envelope antibodies, and subsequently Tat was immunodepleted from the medium. Neurons were exposed to virus/gp120-immunoadsorbed medium with and without immunodepletion of Tat. Neuronal apoptosis was significantly reduced upon immunodepletion of Tat from HIV-infected MDM medium (Figure 5A).


Clade C HIV-1 isolates circulating in Southern Africa exhibit a greater frequency of dicysteine motif-containing Tat variants than those in Southeast Asia and cause increased neurovirulence.

Rao VR, Neogi U, Talboom JS, Padilla L, Rahman M, Fritz-French C, Gonzalez-Ramirez S, Verma A, Wood C, Ruprecht RM, Ranga U, Azim T, Joska J, Eugenin E, Shet A, Bimonte-Nelson H, Tyor WR, Prasad VR - Retrovirology (2013)

Effect of HIV-infected MDM supernatants on neuronal apoptosis. A. HIV-1 infected MDM and uninfected MDM supernatant were diluted with 100 μl of neurobasal medium and incubated with primary human neurons in Matek plates for 18 hours. HIV-1BADA and HIV-1C1084i treatments lead to a greater loss of neuronal viability when compared to HIV-1CIndieC1 and uninfected supernatant. B. Tunnel stained primary human neurons co-stained with anti-neurotubulin antibodies and DAPI (Prolong Gold antifader with DAPI, Invitrogen). The four panels show representative fields for neurons treated with HIV-1BADA, HIV-1C1084i, HIV-1CIndieC1 and media from uninfected macrophages from left to right respectively. The individual (or all) channels are indicated at left. * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3686704&req=5

Figure 4: Effect of HIV-infected MDM supernatants on neuronal apoptosis. A. HIV-1 infected MDM and uninfected MDM supernatant were diluted with 100 μl of neurobasal medium and incubated with primary human neurons in Matek plates for 18 hours. HIV-1BADA and HIV-1C1084i treatments lead to a greater loss of neuronal viability when compared to HIV-1CIndieC1 and uninfected supernatant. B. Tunnel stained primary human neurons co-stained with anti-neurotubulin antibodies and DAPI (Prolong Gold antifader with DAPI, Invitrogen). The four panels show representative fields for neurons treated with HIV-1BADA, HIV-1C1084i, HIV-1CIndieC1 and media from uninfected macrophages from left to right respectively. The individual (or all) channels are indicated at left. * p < 0.05.
Mentions: We next examined the neurotoxicity of HIV-1C1084i Tat when compared to HIV-1IndieC1 TatC and TatB in vitro. Primary human neurons were treated with conditioned medium obtained from HIV-infected MDM and neuronal viability was quantified by using TUNEL assay. Immunofluorescence was used to specifically detect neurons (via staining for neurotubulin), apoptotic cells (TUNEL staining) and nuclei (DAPI staining). The neuronal viability was reduced by 60% when HIV-1C1084i–infected MDM supernatants were used and greater than 80% when HIV-1BADA–infected MDM supernatants were used (Figure 4A and 4B). Media from uninfected Macrophages and HIV-1CIndieC1-infected MDMs resulted in 12% to 25% cell death respectively (Figure 4A, 4B). In order to determine the relative contribution of Tat to neurotoxicity, the MDM supernatants were first depleted of virions and free gp120 using broadly neutralizing envelope antibodies, and subsequently Tat was immunodepleted from the medium. Neurons were exposed to virus/gp120-immunoadsorbed medium with and without immunodepletion of Tat. Neuronal apoptosis was significantly reduced upon immunodepletion of Tat from HIV-infected MDM medium (Figure 5A).

Bottom Line: The proportion of HIV - 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries.In vitro assays revealed that the Southern African isolate, HIV-1C 1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C.Our results indicate that Tat dicysteine motif determines neurovirulence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

ABSTRACT

Background: HIV-1 Clade C (Subtype C; HIV-1C) is responsible for greater than 50% of infections worldwide. Unlike clade B HIV-1 (Subtype B; HIV-1B), which is known to cause HIV associated dementia (HAD) in approximately 15% to 30% of the infected individuals, HIV-1C has been linked with lower prevalence of HAD (0 to 6%) in India and Ethiopia. However, recent studies report a higher prevalence of HAD in South Africa, Zambia and Botswana, where HIV-1C infections predominate. Therefore, we examined whether Southern African HIV-1C is genetically distinct and investigated its neurovirulence. HIV-1 Tat protein is a viral determinant of neurocognitive dysfunction. Therefore, we focused our study on the variations seen in tat gene and its contribution to HIV associated neuropathogenesis.

Results: A phylogenetic analysis of tat sequences of Southern African (South Africa and Zambia) HIV isolates with those from the geographically distant Southeast Asian (India and Bangladesh) isolates revealed that Southern African tat sequences are distinct from Southeast Asian isolates. The proportion of HIV - 1C variants with an intact dicysteine motif in Tat protein (C30C31) was significantly higher in the Southern African countries compared to Southeast Asia and broadly paralleled the high incidence of HAD in these countries. Neuropathogenic potential of a Southern African HIV-1C isolate (from Zambia; HIV-1C 1084i), a HIV-1C isolate (HIV-1 IndieC1) from Southeast Asia and a HIV-1B isolate (HIV-1 ADA) from the US were tested using in vitro assays to measure neurovirulence and a SCID mouse HIV encephalitis model to measure cognitive deficits. In vitro assays revealed that the Southern African isolate, HIV-1C 1084i exhibited increased monocyte chemotaxis and greater neurotoxicity compared to Southeast Asian HIV-1C. In neurocognitive tests, SCID mice injected with MDM infected with Southern African HIV-1C 1084i showed greater cognitive dysfunction similar to HIV-1B but much higher than those exposed to Southeast Asian HIV - 1C.

Conclusions: We report here, for the first time, that HIV-1C from Southern African countries is genetically distinct from Southeast Asian HIV-1C and that it exhibits a high frequency of variants with dicysteine motif in a key neurotoxic HIV protein, Tat. Our results indicate that Tat dicysteine motif determines neurovirulence. If confirmed in population studies, it may be possible to predict neurocognitive outcomes of individuals infected with HIV-1C by genotyping Tat.

Show MeSH
Related in: MedlinePlus