1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.5 Structure of HIV-1
The virions of HIV-1 are spherical in morphology and are between 100-120 nm in diameter (41). The following section describes the: genomic organization of HIV-1, HIV-1 proteins and their function and the structure of HIV-1.
1.5.1 The genomic organization of HIV-1
The HIV-1 genome consists of approximately 9,700 base pairs (bp) divided into nine overlapping genes which are flanked on both the 5’ and 3’ ends by identical long terminal repeats (LTR, 634 bp) (42) (41). The 5’ LTR contains the HIV-1 promoter sequence located in the U3 region which initiates viral transcription whilst the 3’ LTR contains the polyadenylation signal required for dimerization and genome packaging (Figure 1.4a) (43, 44).
The nine genes of HIV-1 encode for three polyproteins and six smaller accessory/regulatory proteins (Figure 1.4a):
group specific antigen (gag, 1,503bp) which encodes for structural components of the virion,
polymerase (pol, 3,012 bp), responsible for encoding the PR, reverse transcriptase (RT) and integrase (INT) enzymes which are encapsulated within the virion,
envelope (Env, 2,571bp), which encodes for viral envelope glycoproteins (gp) expressed in the outer membrane envelope of the virion,
transactivator of transcription factor (Tat, 306 bp) and regulator of virion protein (Rev, 351 bp) which code for regulatory proteins and
viral infectivity factor (Vif, 579 bp), viral protein R (Vpr, 292 bp), viral protein U (Vpu, 249 bp) and the negative regulation factor (Nef, 621 bp) which encode for accessory proteins.
1.5.2 HIV-1 proteins and their function
Gag, Pol and Env, the three proteins shared by all retroviruses, are initially synthesized as polyprotein precursors. These polyprotein precursors require enzymatic cleavage in order to produce mature proteins (Figure 1.4a) (42).
9 1.5.2.1 Group specific antigen (Gag)
The Gag polyprotein precursor named precursor of 55 kDa (Pr55Gag) is cleaved by viral Protease during maturation into p17 matrix (132 amino acids [aa]), p24 capsid (231 aa), p7 nucleocapsid (55 aa), protein of 6 kDA (52 aa, p6) protein of 1 kDA (14 aa, p1) and protein of 2 kDA (16 aa, p2) (Figure 1.4) (42, 44-47). The p17 protein is responsible for: directing the Gag and Gag-Pol polyprotein precursors to the plasma membrane for virion assembly and for the incorporation of envelope glycoproteins into developing virions (48). The p24 protein forms a cone-shaped structure which protects and encapsulates the viral genetic material and facilitates its delivery into the nucleus of the host cell (49) whilst p7 encapsulates unspliced genetic material and has a nucleic acid chaperoning function in which it facilitates the structural rearrangement of genomic material during replication by RT (50). The p6 protein mediates the virus ESCRT (endosomal sorting complex required for transport) dependent budding via two late assembly domains (L-domains) and enables the integration of Vpr into virions (51).
1.5.2.2 Polymerase (Pol)
The Pol enzymes namely PR (99 aa), RT (560 aa) and INT (288 aa) are cleaved from the 160 kDa Gag-pol polyprotein precursor by viral PR. The homodimeric PR is responsible for initiating viral maturation (52, 53). The heterodimer RT converts viral single stranded ribonucleic acid (ssRNA) into double stranded deoxyribose nucleic acid (dsDNA) after viral entry into a cell and has RNAse H activity which facilitates specific degradation of viral RNA from DNA-RNA duplexes (54). Integrase facilitates the incorporation of viral DNA into the host chromosomal DNA (55).
1.5.2.3 Envelope (Env)
The Env polyprotein precursor is cleaved by furin-like host cellular Protease into transmembrane glycoprotein 41 (gp41; 345 aa) and surface glycoprotein 120 (gp120; 511 aa) subunits (56). The gp41 traverses the lipid bilayer and is non-covalently bound to gp120. The gp120 mediates virus attachment to host cells whilst gp41 facilitates entry of the virus into the host cell via fusion of host and viral cellular membranes (Figure 1.4) (57)
10
1.5.2.4 Transactivator of transcription factor (Tat)
Transactivator of transcription factor is a RNA binding protein which recognizes and binds to a transactivator response element (TAR) sequence from the HIV-1 RNA molecule and activates transcription from the viral LTR promoter (58-60).
1.5.2.5 Regulator of virion protein (Rev)
Regulator of virion protein is a regulatory protein which binds to the Rev response element (RRE), a viral RNA element present on individual unspliced or partially spliced viral RNA molecules and initiates the transport of viral RNA transcripts (both spliced and unspliced) out of the nucleus and into the cytoplasm where they serve as transcripts for translation (61, 62).
1.5.2.6 Viral infectivity factor (Vif)
Viral infectivity factor is an accessory protein with a key role in increasing pathogenicity of HIV-1 virions. Its principal target is Apolipoprotein B mRNA-editing enzyme- catalyticpolypeptide-like 3G (APOBEC3G), a member of the APOBEC family of deoxycytidine deaminases which function to suppress viral replication by inducing G to A hyper-mutations within newly synthesized viral DNA thereby inactivating the virus. Viral infectivity factor binds to APOBEC3G and coordinates its proteosomal degradation thereby inhibiting the packaging of APOBEC3G into budding virions (63, 64).
1.5.2.7 Viral protein R (Vpr)
Viral protein R is a second accessory protein which functions primarily to improve viral pathogenicity via: enhancing LTR transcription within infected cells, orchestrating the import of the reverse transcription complex (RTC)4 into the host cell nucleus and inducing cell cycle arrest. It is also involved in inducing apoptosis of T-lymphocytes (65-67).
4 Reverse transcription complex is a term used to describe the infectious viral unit, within which viral ribonucleoprotein enters the host cell and begins reverse transcription of the viral RNA genome (98).
11
1.5.2.8 Viral protein U (Vpu) and Negative regulation factor (Nef)
Viral protein U and Nef are two additional accessory proteins which also contribute towards improving viral pathogenicity. Viral protein U serves to: promote the release of viral progeny from infected cells, downregulate CD4+ cells via the ubiquitin proteasome pathway and downregulate CD155 and natural killer cell receptors in order to evade natural killer cell (NKC) mediated immune responses (68, 69). The main function of the Nef protein is to reduce CD4 and major histocompatibility complex I and II (MHCI and MHCII) cell surface receptors thus facilitating immune evasion (70). Additionally, Nef has a role in the inhibition of apoptosis of infected cells thereby maintaining the longevity of infected cells and contributing to viral propagation and survival (70, 71).
1.5.3 Structure of HIV-1
Each HIV-1 viral particle is surrounded by a lipoprotein rich membrane (i.e. lipid bilayer) with heterodimer complexes comprised of trimers of surface gp120 (which protrudes from the lipid bilayer to the external region of the virion) and transmembrane gp41(which spans the interior of the lipid bilayer) bound together (41, 72). In general, virions have between 14-74 trimers (73). The matrix (MA) protein is attached to the inner surface of the viral lipoprotein membrane. The inner core of the virus is enclosed by the capsid (CA) protein arranged in a fullerene conical structure (49, 74, 75). This structure has approximately 250 CA hexameric rings arranged in a lattice and 12 pentamer rings (5 at the top end and 7 at the bottom end) which provide its conical fullerene structure (76, 77). It encases the: viral genetic material (i.e. a positive sense ssRNA) which is in contact with the nucleocapsid (NC), viral proteins (i.e. p6, Vpr, Vif and Nef), viral enzymes (i.e. PR, RT and INT) and cellular proteins (i.e. APOBEC3G and cyclophilin A [cypA]) (Figure 1.4b) (78-80).