Increased Human Immunodeficiency Virus Type 1 (HIV
http://www.100md.com
病菌学杂志 2005年第16期
UNC Center for AIDS Research
Curriculum in Genetics and Molecular Biology
Departments of Medicine Microbiology and Immunology Biochemistry and Biophysics
Neurology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) surface Env protein has been implicated in the development of HIV-1-associated dementia (HAD). HIV-1 env diversity was analyzed by heteroduplex tracking assay in 27 infected subjects with various neurological statuses. env compartmentalization between the blood and cerebral spinal fluid (CSF) was apparent with all neurological categories. However, in subjects with HAD, significantly more CSF virus was represented by CNS-unique env variants. Variants specialized for replication in the CNS may play a larger role in the development of HAD. Alternatively, HAD may be associated with a more pronounced state of immunosuppression that permits more extensive replication and independent evolution within the CNS compartment.
TEXT
Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) develops within a subset of HIV-1-infected subjects (16, 19, 30). HAD incidence has decreased with the advent of highly active antiretroviral therapy; however, HAD prevalence has increased with longevity (16, 19, 30). HAD is characterized by severe cognitive dysfunction and is generally combined with motor decline. HIV-1-infected patients without dementia frequently have impaired neurological performance in comparison to matched controls. This condition has been termed minor cognitive motor disorder (MCMD) (22, 24). The relationship between MCMD and HAD is unclear.
HIV-1 is present in the central nervous system (CNS) during all stages of disease (4, 10, 11). Essentially identical viral populations are detected in the blood plasma (BP) and cerebral spinal fluid (CSF) within a month of infection (29). The presence of distinct neuropathogenic virus in the CNS is one possible cause of MCMD and HAD. Distinct variants of HIV-1 can replicate in the CNS (1, 3, 7, 8, 12, 14, 15, 20, 21, 35-37, 39), and HIV-1 env variants have been implicated in HAD pathology (23). In this study, env compartmentalization of the blood and CSF was compared among subjects with HAD, MCMD, or no neurological deficits. Viral diversities were measured within the V1/V2 and V3 domains of env by heteroduplex tracking assay (HTA) (5, 6).
Studies of env diversity have predominantly employed cloning to detect major differences between virus in the periphery and virus in the CNS (1-3, 9, 14, 15, 17, 20, 21, 27, 32, 33, 37, 38, 40). HTA differs notably from cloning in that a large number of viral genotypes can be sampled at the same time. The quality of viral population sampling can easily be verified by reproducing the complex HTA banding patterns. All reverse transcriptase (RT)-PCRs and HTA reactions were performed in duplicate to validate the quality of sampling in our analyses. Examples of the reproducibility of sampling are shown in panels A of Fig. 1 and 2 and in Table 1.
env diversity in 27 HIV-1-infected subjects was assessed. Subjects were not receiving therapy at the time that blood and CSF were collected unless otherwise noted. Subject characteristics are described in Table 1. Viral variants from matched BP and CSF samples were amplified by RT-PCR with primers for the V1/V2 or V3 region of env (13, 18). Resulting amplification products were annealed to a strand-specific, radioactive DNA probe representing the region amplified (13, 18). Heteroduplexes were resolved by electrophoresis in a nondenaturing polyacrylamide gel followed by exposure to X-ray film.
Distinct viral genotypes are resolved when sequence differences (insertions, deletions, and clustered mismatches) between the probe and target sequence alter the conformation of the DNA duplex. Various heteroduplex conformations migrate differently through the gel, resulting in their resolution as distinct bands in the gel. A sequence difference (without length differences) of 1 to 2% between the target and the probe is typically sufficient to see a shift (5). Resolution between variants with small sequence differences can be enhanced by the use of heterologous probes containing scattered sequence and length differences relative to the target (6, 13, 18, 26). The HTA is useful in identifying and quantitating both major and minor variants (28). The relative abundance of each variant was analyzed by using phosphorimager technology. In this manner, the viral populations in these compartments were compared.
Compartmentalization between the BP and CSF was clearly evident in the V1/V2 region for the majority of subjects (Fig. 1). A sequence was considered compartmentalized if it was detected in only one compartment (unique) or if shared variants were present in differing relative abundances (enriched) between compartments. Subjects fell into one of the following five compartmentalization categories: (i) no difference in either the variants present or their relative abundances in each compartment (6/22 subjects) (Fig. 1B), (ii) no difference in the identities of variants but shared variants enriched in either BP or CSF (2/22 subjects) (Fig. 1C), (iii) unique variants detected only in the BP (1/22 subjects) (Fig. 1D), (iv) unique variants detected only in the CSF (7/22 subjects) (Fig. 1E), and (v) unique variants detected in both compartments (6/22 subjects) (Fig. 1F). Unique CSF variants, presumably from CNS sources, were detected in 13/22 subjects (Fig. 1E and F).
The V3 region was less diverse and less compartmentalized than the V1/V2 region (Fig. 2). The same five categories of compartmentalization seen in the V1/V2 region were repeated in the V3 region. However, the majority of subjects (12/24) showed no difference in the identity or relative abundance of variants found in the BP and CSF (Fig. 2B). Identical variants were present but enriched in one compartment in 3/24 subjects (Fig. 2C). Unique variants were detected in the BP of 3/24 subjects (Fig. 2D) and in the CSF of 5/24 subjects (Fig. 2E). One subject had a detectable unique variant in each compartment (Fig. 2F). V3 sequence analysis of the majority of variants from 22/24 subjects showed almost exclusive use of the CCR5 coreceptor. An X4 variant was shared between the BP and CSF of two subjects, MCMD subject 301 and HAD subject 2778 (Fig. 2C and E). While it is clear that CXCR4 usage is not required for the development of HAD, further work is needed to assess whether the presence of CXCR4-using variants in the CNS affects progression to HAD.
Shannon entropy [p(i)logp(i)] (31) was calculated to assess overall diversity in V1/V2 and V3. The relative abundance of each band [p(i)] and the number of bands (i [i = 1, ... n, where n is the number of bands]) within a compartment determines the level of entropy. Subjects with many bands with an equal relative abundance would have a high entropy value. A subject with a single variant would have an entropy value of 0. Entropies were not different between compartments within a neurological group or within the same compartment between neurological groups. However, in subjects with HAD, a significantly greater percent of the CSF viral RNA represented CNS-unique variants (P = 0.01, Wilcoxon rank sum test). CNS-unique variants contributed 40 to 96% (median, 62%) of the CSF virus in subjects with HAD, as opposed to 5 to 43% (median, 25%) in subjects without HAD (Table 1). The inability to detect CSF-unique variants in the blood was not the result of bias due to poor sampling; blood RT-PCR amplifications contained a greater number of viral RNA templates than their matched CSF reactions in all subjects except four (one with HAD, two with MCMD, and one with no neurological dysfunction).
Subjects with AIDS had significantly lower CD4 T-cell counts (P = 0.001, Wilcoxon rank sum test). Subjects with HAD had significantly lower CD4 T-cell counts than subjects without dementia (P = 0.01, Wilcoxon rank sum test). Among subjects with AIDS, HAD subjects had significantly lower CD4 counts (P = 0.045, Wilcoxon rank sum test). The difference in CD4 T-cell counts between subjects with MCMD and subjects with no neurological symptoms was not significant (P = 0.7, Wilcoxon rank sum test). Subjects with HAD did not have significantly higher CSF viral loads (P = 0.07, Wilcoxon rank sum test) or BP viral loads (P = 0.16, Wilcoxon rank sum test) than subjects without dementia.
In summary, compartmentalized env variants, either unique or enriched, were readily detected in the CSF. However, the appearance of compartmentalized variants in either the V1/V2 or V3 region did not correlate with neurological disease status. Furthermore, overall diversity was not correlated with neurological disease status. However, subjects with HAD had a greater percentage of CSF virus representing CNS-unique viral variants than subjects without HAD. Previous work suggests that as neurological disease progresses, the viral load source in the CSF becomes more CNS source dependent (7, 8, 34). More specifically, we found that CNS-unique variants, not variants also detected in the periphery, are significantly increased in the CSF during HAD. Increased representation of CNS variants in the CSF may be due to decreased movement of CD4 T-cells between the periphery and CSF, as proposed by Ellis et al. and Price et al. (8, 25). Subjects with HAD that were in our cohort had significantly lower CD4 counts. Pronounced immunological failure may also lead to increased replication and, thus, outgrowth of CNS-unique variants that are then shed into the CSF. It is unclear whether increased replication of CNS-unique variants and/or a novel phenotype of those variants is involved in the development of HAD.
ACKNOWLEDGMENTS
This study was supported by National Institutes of Health grants R01-MH62690, R01-MH067751, and GCRC-RR00046 and the University of North Carolina Center for AIDS Research (P30-AI50410). K.R. was supported in part by National Institutes of Health training grant T32-AI007419.
We thank Laura Lapkauskaite (Department of Biostatistics, University of North Carolina—Chapel Hill) for her assistance with the statistical analysis.
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Curriculum in Genetics and Molecular Biology
Departments of Medicine Microbiology and Immunology Biochemistry and Biophysics
Neurology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) surface Env protein has been implicated in the development of HIV-1-associated dementia (HAD). HIV-1 env diversity was analyzed by heteroduplex tracking assay in 27 infected subjects with various neurological statuses. env compartmentalization between the blood and cerebral spinal fluid (CSF) was apparent with all neurological categories. However, in subjects with HAD, significantly more CSF virus was represented by CNS-unique env variants. Variants specialized for replication in the CNS may play a larger role in the development of HAD. Alternatively, HAD may be associated with a more pronounced state of immunosuppression that permits more extensive replication and independent evolution within the CNS compartment.
TEXT
Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) develops within a subset of HIV-1-infected subjects (16, 19, 30). HAD incidence has decreased with the advent of highly active antiretroviral therapy; however, HAD prevalence has increased with longevity (16, 19, 30). HAD is characterized by severe cognitive dysfunction and is generally combined with motor decline. HIV-1-infected patients without dementia frequently have impaired neurological performance in comparison to matched controls. This condition has been termed minor cognitive motor disorder (MCMD) (22, 24). The relationship between MCMD and HAD is unclear.
HIV-1 is present in the central nervous system (CNS) during all stages of disease (4, 10, 11). Essentially identical viral populations are detected in the blood plasma (BP) and cerebral spinal fluid (CSF) within a month of infection (29). The presence of distinct neuropathogenic virus in the CNS is one possible cause of MCMD and HAD. Distinct variants of HIV-1 can replicate in the CNS (1, 3, 7, 8, 12, 14, 15, 20, 21, 35-37, 39), and HIV-1 env variants have been implicated in HAD pathology (23). In this study, env compartmentalization of the blood and CSF was compared among subjects with HAD, MCMD, or no neurological deficits. Viral diversities were measured within the V1/V2 and V3 domains of env by heteroduplex tracking assay (HTA) (5, 6).
Studies of env diversity have predominantly employed cloning to detect major differences between virus in the periphery and virus in the CNS (1-3, 9, 14, 15, 17, 20, 21, 27, 32, 33, 37, 38, 40). HTA differs notably from cloning in that a large number of viral genotypes can be sampled at the same time. The quality of viral population sampling can easily be verified by reproducing the complex HTA banding patterns. All reverse transcriptase (RT)-PCRs and HTA reactions were performed in duplicate to validate the quality of sampling in our analyses. Examples of the reproducibility of sampling are shown in panels A of Fig. 1 and 2 and in Table 1.
env diversity in 27 HIV-1-infected subjects was assessed. Subjects were not receiving therapy at the time that blood and CSF were collected unless otherwise noted. Subject characteristics are described in Table 1. Viral variants from matched BP and CSF samples were amplified by RT-PCR with primers for the V1/V2 or V3 region of env (13, 18). Resulting amplification products were annealed to a strand-specific, radioactive DNA probe representing the region amplified (13, 18). Heteroduplexes were resolved by electrophoresis in a nondenaturing polyacrylamide gel followed by exposure to X-ray film.
Distinct viral genotypes are resolved when sequence differences (insertions, deletions, and clustered mismatches) between the probe and target sequence alter the conformation of the DNA duplex. Various heteroduplex conformations migrate differently through the gel, resulting in their resolution as distinct bands in the gel. A sequence difference (without length differences) of 1 to 2% between the target and the probe is typically sufficient to see a shift (5). Resolution between variants with small sequence differences can be enhanced by the use of heterologous probes containing scattered sequence and length differences relative to the target (6, 13, 18, 26). The HTA is useful in identifying and quantitating both major and minor variants (28). The relative abundance of each variant was analyzed by using phosphorimager technology. In this manner, the viral populations in these compartments were compared.
Compartmentalization between the BP and CSF was clearly evident in the V1/V2 region for the majority of subjects (Fig. 1). A sequence was considered compartmentalized if it was detected in only one compartment (unique) or if shared variants were present in differing relative abundances (enriched) between compartments. Subjects fell into one of the following five compartmentalization categories: (i) no difference in either the variants present or their relative abundances in each compartment (6/22 subjects) (Fig. 1B), (ii) no difference in the identities of variants but shared variants enriched in either BP or CSF (2/22 subjects) (Fig. 1C), (iii) unique variants detected only in the BP (1/22 subjects) (Fig. 1D), (iv) unique variants detected only in the CSF (7/22 subjects) (Fig. 1E), and (v) unique variants detected in both compartments (6/22 subjects) (Fig. 1F). Unique CSF variants, presumably from CNS sources, were detected in 13/22 subjects (Fig. 1E and F).
The V3 region was less diverse and less compartmentalized than the V1/V2 region (Fig. 2). The same five categories of compartmentalization seen in the V1/V2 region were repeated in the V3 region. However, the majority of subjects (12/24) showed no difference in the identity or relative abundance of variants found in the BP and CSF (Fig. 2B). Identical variants were present but enriched in one compartment in 3/24 subjects (Fig. 2C). Unique variants were detected in the BP of 3/24 subjects (Fig. 2D) and in the CSF of 5/24 subjects (Fig. 2E). One subject had a detectable unique variant in each compartment (Fig. 2F). V3 sequence analysis of the majority of variants from 22/24 subjects showed almost exclusive use of the CCR5 coreceptor. An X4 variant was shared between the BP and CSF of two subjects, MCMD subject 301 and HAD subject 2778 (Fig. 2C and E). While it is clear that CXCR4 usage is not required for the development of HAD, further work is needed to assess whether the presence of CXCR4-using variants in the CNS affects progression to HAD.
Shannon entropy [p(i)logp(i)] (31) was calculated to assess overall diversity in V1/V2 and V3. The relative abundance of each band [p(i)] and the number of bands (i [i = 1, ... n, where n is the number of bands]) within a compartment determines the level of entropy. Subjects with many bands with an equal relative abundance would have a high entropy value. A subject with a single variant would have an entropy value of 0. Entropies were not different between compartments within a neurological group or within the same compartment between neurological groups. However, in subjects with HAD, a significantly greater percent of the CSF viral RNA represented CNS-unique variants (P = 0.01, Wilcoxon rank sum test). CNS-unique variants contributed 40 to 96% (median, 62%) of the CSF virus in subjects with HAD, as opposed to 5 to 43% (median, 25%) in subjects without HAD (Table 1). The inability to detect CSF-unique variants in the blood was not the result of bias due to poor sampling; blood RT-PCR amplifications contained a greater number of viral RNA templates than their matched CSF reactions in all subjects except four (one with HAD, two with MCMD, and one with no neurological dysfunction).
Subjects with AIDS had significantly lower CD4 T-cell counts (P = 0.001, Wilcoxon rank sum test). Subjects with HAD had significantly lower CD4 T-cell counts than subjects without dementia (P = 0.01, Wilcoxon rank sum test). Among subjects with AIDS, HAD subjects had significantly lower CD4 counts (P = 0.045, Wilcoxon rank sum test). The difference in CD4 T-cell counts between subjects with MCMD and subjects with no neurological symptoms was not significant (P = 0.7, Wilcoxon rank sum test). Subjects with HAD did not have significantly higher CSF viral loads (P = 0.07, Wilcoxon rank sum test) or BP viral loads (P = 0.16, Wilcoxon rank sum test) than subjects without dementia.
In summary, compartmentalized env variants, either unique or enriched, were readily detected in the CSF. However, the appearance of compartmentalized variants in either the V1/V2 or V3 region did not correlate with neurological disease status. Furthermore, overall diversity was not correlated with neurological disease status. However, subjects with HAD had a greater percentage of CSF virus representing CNS-unique viral variants than subjects without HAD. Previous work suggests that as neurological disease progresses, the viral load source in the CSF becomes more CNS source dependent (7, 8, 34). More specifically, we found that CNS-unique variants, not variants also detected in the periphery, are significantly increased in the CSF during HAD. Increased representation of CNS variants in the CSF may be due to decreased movement of CD4 T-cells between the periphery and CSF, as proposed by Ellis et al. and Price et al. (8, 25). Subjects with HAD that were in our cohort had significantly lower CD4 counts. Pronounced immunological failure may also lead to increased replication and, thus, outgrowth of CNS-unique variants that are then shed into the CSF. It is unclear whether increased replication of CNS-unique variants and/or a novel phenotype of those variants is involved in the development of HAD.
ACKNOWLEDGMENTS
This study was supported by National Institutes of Health grants R01-MH62690, R01-MH067751, and GCRC-RR00046 and the University of North Carolina Center for AIDS Research (P30-AI50410). K.R. was supported in part by National Institutes of Health training grant T32-AI007419.
We thank Laura Lapkauskaite (Department of Biostatistics, University of North Carolina—Chapel Hill) for her assistance with the statistical analysis.
REFERENCES
Babas, T., D. Munoz, J. L. Mankowski, P. M. Tarwater, J. E. Clements, and M. C. Zink. 2003. Role of microglial cells in selective replication of simian immunodeficiency virus genotypes in the brain. J. Virol. 77:208-216.
Chang, J., R. Jozwiak, B. Wang, T. Ng, Y. C. Ge, W. Bolton, D. E. Dwyer, C. Randle, R. Osborn, A. L. Cunningham, and N. K. Saksena. 1998. Unique HIV type 1 V3 region sequences derived from six different regions of brain: region-specific evolution within host-determined quasispecies. AIDS Res. Hum. Retrovir. 14:25-30.
Chen, H., C. Wood, and C. K. Petito. 2000. Comparisons of HIV-1 viral sequences in brain, choroid plexus and spleen: potential role of choroid plexus in the pathogenesis of HIV encephalitis. J. Neurovirol. 6:498-506.
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