In this article we will discuss about:- 1. Structure of HIV 2. Genome Organization of HIV-1 3. Steps of Entry 4. Replication 5. Assembly.
Structure of HIV:
HIV is different in structure from other retroviruses. HIV is of 100-120 nm diameters containing an protein envelope to which spicules of glycoprotein are attached. The envelope encloses an icosahedral capsid core that possesses identical macromolecules of RNA as genetic material. Three dimensional structure of the viral envelope appears like a sphere made up from an assembly of 12 pentamers and 20 hexamers (Fig. 17.41).
The capsid is conical and composed of 2,000 copies of the viral protein p24. The two copies of positive single-stranded RNA are tightly bound with nucleocapsid proteins, p7 and enzymes such as reverse transcriptase, proteases, ribonuclease and integrase. These are required for the development of the virion. Matrix is composed of viral protein p17 which surrounds the capsid. Hence, the integrity of the virion particle it maintained.
In turn, the matrix is surrounded by the viral envelope which is composed of two layers of phospholipids of host cell which originates at the time of budding from the cell of newly formed virus particles. The host cell proteins and about 70 copies of a complex HIV proteins are embedded in the viral envelope that protrude through the surface of the virus particle. This protein is called Env which constitutes spikes.
It consists of a cap which is made up of three molecules of glycoprotein (gp) 120, and a stem of three gp41 molecules that anchor the spike into the viral envelope. The virus attaches to host cell with glycoprotein and gets fused with target cells to initiate the infectious cycle. Both of these surface proteins (especially gp 120) have been considered as targets of future treatments or vaccines against HIV.
Genome Organization of HIV-1:
The RNA genome consists of at least 7 structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and tev) encoding 19 proteins (Fig. 17.42). The open reading frames for various polypeptides are shown as rectangles.
Multiple-spliced mRNA transcripts encoding various proteins are shown with splice-sites together with 5′-cap and 3′ poly A tails. Major translated polypeptides from these mRNAs are initially processed to produce 15 protein molecules. In addition, there is a third type of nucleic acid present in all particles which is a special type of tRNA (usually trp, pro or. lys). It is required for replication.
Three of the nine genes (i.e. gag, pol, and env) contain the information which is required to synthesise the structural proteins for new virus particles. For example, env codes for a protein called gpl60 which, after getting broken by a viral enzyme, forms gp120 and gp41. Structure of different regions is given in Fig. 17.43.
Structure and function of these regions are given as below:
i. R Region:
It is a short (18-250 nucleotides) sequence which forms a direct repeat at both ends of the genome; therefore, it is ‘terminally redundant’. The R region is present at both the ends.
ii. U5:
It is a unique, non-coding region of 75-250 nucleotides which is the first part of the genome to be reverse transcribed forming the 3′ end of the provirus genome.
iii. Primer Binding Site (PBS):
It is 18 nucleotide-long and complementary to the 3′ end of the specific tRNA primer used by the virus to begin reverse transcription.
iv. Leader:
It is a relatively long (90-500 nucleotides) non-translated region downstream of the transcription start site; therefore it is present at the 5′ end of all virus mRNAs.
v. Polypurine Tract (PPT):
It is a short (-10 nucleotides) run of A/G residues which is responsible for initiating the synthesis of (-n) strand during reverse transcription.
vi. U3:
It is a unique non-coding region of 200-1,200 nucleotides which forms the 5′ end of the provirus after reverse transcription. It contains promoter elements which are responsible for transcription of the provirus (Fig. 17.44).
The other six genes e.g. tat, rev, nef, vif, vpr, and vpu (of in the case of HFV-2) are the regulatory genes. These genes enc proteins that control the ability of HIV to infect cells, produce virus particles or cause disease. The two Tat proteins (e.g. pi6 pi4) act as the transcriptional trans activators for the LTR promoter.
They activate transcription by binding the TAR RNA element. Rev protein (p19) binds to the RRE RNA element and start shutl of RNAs from the nucleus and the cytoplasm. AP0BEC3G is a protein which deaminates DNA: RNA hybrids and/or interferes with the Pol protein. The Vif protein (p23) prevents the action of APOBEC3G.
The Vpr protein (P14) inhibits cell division at G2/M stage. CD4 as well as the MHC class I and class It molecules. The Vpu protein (p16) helps the release of newly synthesised virus particles from infected cells. The ends of each strand of HIV RNA contain a sequence of repeated nucleotides called ‘R0region’ or the ‘long terminal repeat’ (LTR).
Regions in the LTR act as switches to control production of new viruses. The LTR regions can be triggered by proteins from either HIV or the host cell. The Gag and Rev Proteins recognize the Psi element which is involved in viral genome packaging. The in the Gag-Pol reading frame required to make functional Pol. Frame shifting is carried out by the SLIP element (TTTTTT).
Steps of Entry of HIV:
The infectious particle first binds to target cells using semi-or non-specific interactions between the viral envelope and cell surface glycans or adhesion factors. Then the envelope glycoprotein gpl20 interacts with the CD4 antigen. This initiates a conformational change in gpl20 that facilitates its binding to a co-receptor molecule (Fig. 17.45).
Further conformational changes in the gpl20-gp41 complex then lead to exposure of the fusion-peptide region of gp41 and its insertion into the host cell membrane. The viral and cell membranes fuse, the viral uncoating is done and viral RNA plus virion proteins enter the cytosol. Within this reverse transcription complex (viral RNA and virion proteins), reverse transcriptase catalyses the synthesis of complementary DNA.
Replication of HIV:
The resulting complex containing viral cDNA (pre-integration complex) is transported to the host cell nucleus where the viral integrase enzyme catalyses integration of viral cDNA within host cell DNA to form the proving (Fig. 17.46).
In some instances, viral cDNA that is translocated to the nucleus circularizes to form episomes containing one or two long terminal repeats (LTR). These circular forms of viral cDNA are dead-end products of viral replication.
The ends of the LTRs consist of inverted repeats of 4-6 bp. These are brought together to form a cleavage site for IN and are cleaved to form a staggered cut. This process is catalysed by TN polypeptide which is a part of the reverse transcriptase complex. Then this molecule is inserted into the host cell DNA.
The net result of the integration process is that:
(i) The integrated provirus contains 1 or 2 less bases at the end of each LTR,
(ii) The ends of the integrated LTRs always have the same sequence: 5′ – TG…CA – 3′ , and
(iii) 4-6 bp of host cell DNA flanking the integrated provirus are duplicated. A staggered cut (5′ overhang) is introduced into both the ends of the LTRs and the host cell DNA, followed by joining of the cut ends and repair of the free 3′ ends.
Integration is a highly specific reaction with respect to the provirus, but random with respect to host cell DNA. Earlier, it was thought that the 2-LTR circle was the substrate for integration, but it is now believed that the linear form (i.e. the direct product of reverse transcription) is the actual substrate used. After integration, the provirus is present for the lifetime of the cell. There is no specific mechanism for excision of the provirus.
Assembly of HIV:
Assembly of capsid/nucleocapsid occurs in the cytoplasm (or at the cell surface) which later on buds out through the cell membrane, acquiring the envelope during this process (Fig. 17.46). If assembly occurs at cell surface, thickened patches begin to form in the membrane (Env proteins on outer surface. Gag proteins inside).
The genome is packaged when the particle has budded out through the membrane. In both types, ‘maturation’ is accomplished with the catalysis and cleavage events by viral protease which results in budding of the particles.
During this process, considerable structural changes occur which results in completely rearrangement of the smooth gag shell of the immature particle that lead to condensation of the core visible in mature particles.