Compartmentalization, Tissue-Specific Selection and Mutation of Envelope V1 Region, Nef and Integrase of Neuropathogenic SIVsmmFGb in the Central Nervous System and Lymph Nodes of Infected Pigtailed Macaques at One Week and Two Months Post- Infection Público

Abstract

The simian lentivirus quasispecies SIVsmmFGb induces neuropathology in over 90% of infected pigtailed macaques and is a reliable model of HIV neuropathogenesis. However, little is understood about the genetic diversity of this virus, development of the quasispecies over the course of infection, whether the virus compartmentalizes between tissues and how these compartments develop over time. Initial seeding of the CNS and lymph node reduced Env V1 region and Int genetic diversity but had a variable effect on Nef. By two mpi, selective pressures affected the diversity of Nef sequences between tissues, but changes to Env V1 region and Int variety were similar across all tissues. At both one wpi and two mpi, env V1 region and int genes formed separate compartments between areas of the CNS, as well as between the CNS regions and the lymph nodes. The nef genes compartmentalized separately between all tissues at one wpi, but no longer segregated between lymph nodes at two mpi. Negative selection on nef and int genes increased in the brain regions and one lymph node over time, while positive selection of env V1 region increased over time in some CNS regions. Convergent evolution of both nef and env V1 region sequences, and divergent evolution of int genes, was noted between compartments. Intra-tissue temporal segregation occurred for all three genes and was stronger in the CNS for env V1 region and nef. Functional differences in int and env V1 region genes decreased between tissues over time, but functional differences in nef sequences increased.

We also analyzed the Env V1 region, Nef and Int amino acid sequences for mutations in important functional domains. The average Env V1 region length and number of O-linked glycosylations, as well as the presence of a six-amino acid insertion, increased over time in the CNS and lymph nodes. For Nef, the prevalence of mutations increased over time in the basic region and N-proximal Y residues, but the frequency of mutations declined in the acidic region. The proportion of mutations in the Nef thioesterase and AP interaction sites increased over time only in the CNS.

Table of Contents

I. Introduction...1

A. Epidemiology of HIV/AIDS
B. HIV-Associated Neurocognitive Disorders
C. HIV-Associated Dementia
D. HAART and HIV-Associated Dementia
E. Lesions in HIV and SIV Neuropathogenesis
F. Pathways of Virus Entry into the CNS
G. Virus Target Cells in the CNS
H. Virus Replication in the CNS
I. Immune Responses to HIV and SIV Infection of the CNS
J. Neurotoxic Viral Factors
K. Neurotoxic Factors Released by Host Cells
L. HIV and SIV Envelope V1 Region
M. HIV and SIV Nef
N. HIV and SIV Integrase
O. The SIV-Macaque Animal Model of HIV Neuropathogenesis
P. SIVsmmFGb Virus Isolation and Characteristics
Q. Lentiviral Compartmentalization

II. Materials and Methods - Adapted from Reeve, et al. ARHR. 2009. 25(6):583-601, Reeve, et al. In Press and Reeve, et al. Unpublished Data...41

A. SIVsmmFGb Stock Virus Isolation and PCR Amplification
B. Pigtailed Macaque Inoculation and SIVsmmFGb-Infected Tissue Harvesting
C. SIVsmmFGb Proviral DNA Extraction and Preparation from Infected Pigtailed Macaque Tissues
D. PCR Amplification of SIVsmmFGb Genes From Tissue-Derived Proviral DNA
E. SIVsmmFGb Gene Cloning Strategy
F. DNA Sequence Analysis
G. SIVsmmFGb Stock Virus Amino Acid Sequence Grouping
H. Tissue-Isolated SIVsmmFGb Viral Amino Acid Sequence Alignment and Grouping
I. Phylogenetic and Phenetic Compartmentalization Analyses of SIVsmmFGb Amino Acid and Nucleotide Sequences
J. Synonymous and Nonsynonymous Substitution Analyses of SIVsmmFGb Nucleotide Sequences
K. Determination of the Average Pairwise Distances of SIVsmmFGb Nucleotide Sequences from Pigtailed Macaque Tissues
L. Calculation of the Prevalence of Amino Acid Mutations in Functional Domains of SIVsmmFGb Env V1 Region, Nef and Int
M. Determination of the Frequency of Amino Acid Changes at Each Position of SIVsmmFGb Env V1 Region, Nef and Int
N. Sequence Data

III. Chapter 1: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection - Adapted from Reeve, et al. ARHR. 2009. 25(6):583-601...53

A. Results

1. SIVsmmFGb Stock Virus Grouping
2. ISH of SIVsmmFGb in Brain and Peripheral Tissues of Infected Pigtailed Macaques
3. Diversity of Env V1 Region, Nef and Int Amino Acid Sequences Isolated from Pigtailed Macaque Brain and Lymph Node Tissues Compared to Stock Virus
4. Phylogenetic Analysis of Compartmentalization of Env V1 Region, Nef and Int Amino Acid Sequences Isolated from Pigtailed Macaque Brain and Lymph Node Tissues
5. Phenetic Analysis of the Compartmentalization of SIVsmmFGb env V1 Region, nef and int Sequences Isolated from Pigtailed Macaque CNS and Lymph Node Tissues
6. dS and dN Values for SIVsmmFGb env V1 Region, nef and int Sequences Isolated from Pigtailed Macaque CNS and Lymph Node Tissues

B. Discussion

IV. Chapter 2: Neuropathogenic SIVsmmFGb Genetic Diversity and Selection-Induced Tissue-Specific Compartmentalization During Chronic Infection and Temporal Evolution of Viral Genes in Lymphoid and Central Nervous System Tissues - Adapted from Reeve, et al. In Press...74

A. Results

1. Diversity of Env V1 Region, Nef and Int Amino Acid Sequences Isolated from Pigtailed Macaque Brain Regions and Lymph Node Tissues at Two Months Post-Infection
2. Phylogenetic Compartmentalization Analysis of Env V1 Region, Nef and Int Amino Acid Sequences Isolated from Pigtailed Macaque Brain Regions and Lymphoid Tissues Two Months Post-Infection
3. Phenetic Compartmentalization Analysis of env V1 Region, nef and int Nucleotide Sequences Isolated from Pigtailed Macaque Brain Regions and Lymphoid Tissues Two Months Post-Infection
4. Synonymous and Nonsynonymous Nucleotide Substitution Rates of SIVsmmFGb env V1 Region, nef and int Sequences Isolated from Pigtailed Macaque Brain Regions and Lymph Node Tissues Two Months Post-Infection
5. Phylogenetic Analysis of Temporal Compartmentalization of Envelope V1 Region, Nef and Integrase Amino Acid Sequences between Pigtailed Macaque Brain and Lymph Node Tissues Harvested at One Week and Two Months Post-Infection

B. Discussion

V. Chapter 3: Pairwise Distances Between SIVsmmFGb env V1 Region, nef and int Nucleotide Sequences and Mutations in Functional Domains of Env V1 Region, Nef and Integrase Amino Acid Sequences from the Central Nervous System and Lymph Nodes of Infected Pigtailed Macaques at One Week and Two Months Post-Infection - Adapted from Reeve, et al. Unpublished Data...93

A. Results

1. Comparison of Pairwise Distances of SIVsmmFGb env V1 Region, nef and int Sequences Between and Within Lymph Node Tissues and CNS Regions from Infected Pigtailed Macaques at One Week and Two Months Post-Infection
2. Prevalence of Sequences with Mutations in Functional Domains of SIVsmmFGb Env V1 Region, Nef and Int Amino Acid Sequences from the Lymph Nodes and Central Nervous System of Infected Pigtailed Macaques at One Week and Two Months Post-Infection
3. Prevalence of Specific Mutations in Functional Domains of SIVsmmFGb Env V1 Region, Nef and Int Amino Acid Sequences in the Central Nervous System and Lymph Nodes of Infected Pigtailed Macaques at One Week and Two Months Post-Infection

B. Discussion

VI. Conclusions...113

VII. Index of Figures...115

VIII. Figures 1 - 41...119

IX. Index of Tables...167

X. Tables 1 - 51...174

XI. References...248

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology Biology, Virology
Palabra Clave	Neuropathogenesis SIVsmmFGb Evolution Compartmentalization
Committee Chair / Thesis Advisor	Novembre, Francis, Emory University
Committee Members	Rota, Paul A, Emory University Grakoui, Arash, Emory University Amara, Rama Rao, Emory University Tyor, William, Emory University

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