CHAPTER 1 INTRODUCTION
1.4 HIV-1 Pathogenesis
1.4.1 Acute infection
14 363) impair virus assembly, but mutations in the capsid N-terminal domain (residues 133- 277) impair maturation [69]. Virus maturation is essential for virus infectivity [70] (Section 1.3.1). Recently, mutation of a conserved Gag residue in p6, 488, was demonstrated to reduce Gag cleavage between the capsid and spacer peptide resulting in irregular core structure and consequently reduced viral infectivity [64]. Refer to Table 1.1 for a summary of the role of Gag in the HIV-1 replication cycle.
Virion maturation completes the viral replication cycle, which requires approximately 2 days in total [1, 71]. In untreated chronic infection, the replication cycle results in the production of approximately 1010 to 1011 virions per day, each with a cell-free half-life of 30 to 60 minutes [1]. Productively infected CD4+ T cells have a half-life of 1 day and are therefore rapidly eliminated [1, 72]. However, the pathogenesis of HIV-1 is complex and the disease is likely the result of both direct cytopathic and indirect effects [73].
Table 1.1 Role of Gag proteins in the HIV-1 replication cycle Gag
protein
Residues Role Replication
step
Matrix (p17):
residues 1- 132
9, 67, 72, 77 Serine residues phosphorylated on viral entry, which may promote membrane dissociation.
Early post-entry steps
21 Mutation increased membrane binding, reducing viral DNA synthesis.
Early post-entry steps
25-32, 110-114 NLSs which may be involved in nuclear import. Nuclear import 26, 27 Impaired integration when mutated. Integration 18, 22 Impaired nuclear export when mutated. Nuclear export 1-100: 6-17, 18,
31, 35, 63
Envelope glycoprotein incorporation. Residues 6-17 bind TIP47, which mediates the incorporation. This interaction couples Gag maturation to fusion.
Assembly/Entry
11-19, 132 Trafficking of Gag proteins. Assembly
85-89 Targeting virus assembly to plasma membrane. Assembly 15-31 Basic domain mediating membrane binding. Assembly 7-9 Hydrophobic domain synergising with basic domain
and myristate to promote membrane binding.
Assembly 1-7 Binding of myristate required for membrane binding. Assembly 12, 89 Regulation of myristate exposure to control
membrane binding.
Assembly
Capsid (p24):
residues 133- 363
308-362 Binds to lysyl-tRNA synthetase that mediates packaging of tRNAlys which is required for initiation of reverse transcription.
Reverse transcription 217-225 Binds cyclophilin A which protects HIV-1 from an
unknown restriction factor and facilitates proper uncoating of the capsid.
Uncoating
135 Trafficking of Gag proteins. Assembly
283-363 Mutations in this domain mainly impair virus assembly.
Assembly 133-277 Mutations in this domain mainly impair virus
maturation.
Maturation/Entry
Nucleocapsid (p7):
residues 378- 432
400, 421 Impaired reverse transcription when mutated. Zn fingers chaperone strand transfers in reverse transcription.
Reverse transcription 384, 387, 388,
391, 400, 421
Involved in the protection of newly synthesised DNA.
Steps following reverse
transcription 400, 421 Mutations impaired 3’ end processing of viral DNA. Integration 390-423 Hydrophobic plateau binding RNA genome to
nucleocapsid. This regulates myristate exposure promoting membrane binding and provides a scaffold for virus assembly.
Assembly
380, 384, 387, 388, 391, 397, 403, 406, 409-411, 415, 418, 424, 429
Basic residues required for budding possibly through interaction with ESCRT machinery.
Budding
p6: residues 449-500
463-466, 482-484, 489-493
Mediates incorporation of Vpr into virions. Assembly 455-458 (PTAP) Interacts with Tsg101protein (part of ESCRT-I) to
recruit ESCRT machinery for budding.
Budding 483-492: 483-485,
489, 492
Interacts with Alix protein to recruit ESCRT machinery for budding.
Budding
488 Mutation disrupts Gag cleavage by protease. Maturation /Entry HIV-1 – human immunodeficiency virus type 1; NLS - nuclear localisation signal; TIP47 – tail-interacting protein of 47 kDa; tRNA – transfer ribonucleic acid; Zn - zinc; DNA – deoxyribonucleic acid; ESCRT - endosomal sorting complexes required for transport; p6 – protein of 6 kDa; Vpr – viral protein R; Tsg101 protein – tumour susceptibility 101 protein;
Alix – apoptosis-linked gene 2-interacting protein X.
16 sequencing of HIV-1 transmitted viruses indicate that the first cells infected are sub- optimally activated CD4+ CCR5+ memory T cells [72, 75, 76] and that the transmitted virus is dependent on CCR5 (not CXCR4) for cell entry [77]. Other cells found in the mucosa that could be infected in the acute phase include Langerhans cells (mucosal dendritic cells) and macrophages [73, 78, 79]. The initial infection is supported by signalling from the mucosal epithelial cells (triggered by pathogen-mediated activation of toll-like receptors [TLRs]) that recruits dendritic cells, which in turn secrete cytokines that attract activated CD4+ T cells susceptible to infection [75, 76]. Thus, the innate immune response promotes establishment of infection (Section 1.4.4.1).
After approximately 10 days (the eclipse phase [72, 80, 81]), the expansion of infection is sufficient to result in dissemination of cell-free virus or infected cells to the draining lymph node and consequently the bloodstream and secondary lymphoid organs [72, 75]. The dendritic cells in the mucosa may also internalise HIV-1 through dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptors and retain the infectious particles, migrate to the draining lymph node, and make contact with CD4+ T cells in the lymph node, resulting in efficient transfer of HIV-1 that assists in dissemination of the virus [1, 73, 79]. Furthermore, trapping of HIV-1 virions by dendritic cells in the lymph node exposes susceptible CD4+ T cells to infection as they migrate into the lymph node follicle to provide help to B cells [1]. Since many susceptible cells are found in close proximity within the lymphoid tissues, a massive burst of HIV-1 replication occurs in these tissues. This is particularly true in the gut-associated lymphoid tissue which harbours a high concentration of activated CD4+ CCR5+ memory T cells [72]. Interestingly, it was recently noted that successfully transmitted viruses have a high affinity for α4β7+ CD4+ T cells, which are present in the vaginal/rectal mucosa and home to the gut (where the exponential
replication is best facilitated), suggesting this characteristic may be crucial to the successful establishment of infection [11]. A massive depletion of CD4+ T cells occurs during this early exponential phase of viral replication. During this replication burst, at around 21-28 days post-infection, peak plasma viremia (>1 million HIV-1 RNA copies/ml) is reached, and more than 50% of individuals may experience acute viral symptoms, such as fever, rash, and/or lymphadenopathy [72, 82].
Prior to this replication burst, HIV-1 RNA is first detectable in the blood (Fiebig stage I, 10- 17 days post-infection) followed by p24 antigen (Fiebig stage II, 17-22 days post-infection) [80, 81]. The first HIV-specific antibodies become detectable (seroconversion) during the phase of peak viremia (Fiebig stage III, 22-25) [80, 81], but they are non-neutralising (i.e.
they are unable to block virus entry into cells) (Section 1.4.4.2). Viremia then declines during the following Fiebig stages (stage IV [25-31 days post-infection], stage V [31-101 days post-infection], and stage VI [open-ended, early chronic infection phase]), which are characterised by the detection of different antibodies by Western blot, and a steady state (viral set point) is established during Fiebig stages V or VI [80, 81]. The CD8+ T cell responses emerge as plasma viremia approaches peak and peaking of these responses is coincident with decline in viremia, suggesting that the suppression of viral replication is largely mediated by CD8+ T cells [72] (Section 1.4.4.3.1).
During the acute phase of infection a reservoir of latently infected cells is established [72, 74]. These cells are primarily resting memory CD4+ T cells [83], but macrophages are also important reservoirs [73, 78]. These latently infected cells are invisible to the immune response, safe from antiretroviral drugs, long-lived (some persist for decades), and produce infectious virus only when re-activated; thereby preventing elimination of the infection [78,
18 83] (Section 1.5.1). Productively infected macrophages are also an important reservoir as they can survive for several weeks and efficiently transmit HIV-1 to CD4+ T cells via cell- to-cell synapses [84].