Definition and Life Cycle of a Retrovirus
Figure 1: a diagrammatic representation of the retroviral life cycle
This family contains enveloped viruses which have single-stranded positive RNA as genome and carry two essential proteins within their capsid, one is a Reverse Transcriptase, necessary to produce a DNA version of the viral genome, and the other an Integrase, enzyme necessary for integration of the viral DNA into the host genome.
As for other viruses, the life cycle of a retrovirus (Figure 1) is characterized by six basic stages: attachment, penetration, uncoating, replication, assembly and finally release. The stages are briefly described below.
In order to attach, retroviral glycoproteins bind to matching receptors on the surface of a cell. Which cells (or tissues) are infected depends on the retroviral “tropism” and this, in turn, is determined by the receptors that the virus can bind to. The viral attachment often occurs through multiple weak interactions between the numerous pairs of macromolecules (glycoproteins).
Penetration follows attachment: virions enter the host cell through receptor-mediated endocytosis or through cell-surface protein-mediated fusion of membranes (see Figure 2 below). In the case of endocytosis, the fusion of the viral membrane occurs within the endosome. The free capsid enters the cytoplasm. The next stage is uncoating of the viral capsid; this may occur through simple dissociation or through degradation by viral or host enzymes. The result is the release of the positive RNA genome into the host cell. Now, the specific processes happen (Reverse Transcription and Integration) giving rise to the double-stranded DNA which will be incorporated in the cell’s genome at random locations. At this moment, the retrovirus changes its’ name and becomes a provirus.
The provirus can stay dormant in the infected cell, or the cell may immediately initiate the process of viral propagation and start to transcribe the proviral DNA into mRNA thanks to the host RNA-polymerase. Following transcription, the host ribosomes translate the mRNA into the precursor proteins which are further processes into mature proteins. In fact, this step will make new viral enzymes and capsid proteins, while the viral RNA will be made in the nucleus.
These pieces are then gathered together and assembled into new virus particles, which will be pinched off of the cell membrane (budding) as viruses (release).
Focus on attachment and penetration: the entry of HIV into the cell and the role of CCR5 protein
The mechanism that we will explain involves the viral envelope, with the glycoproteins gp120 and gp41, and the host-cell membrane, with CD4 and CCR5 proteins (named receptor and co-receptor respectively). Gp41, which is situated in the envelope membrane, binds gp120, a protein with three variable loops. When the two membranes are in close proximity, variable loop 3 of gp120 will bind to a CD4 receptor (see Figure 2 below). This brings a change in the conformation of the gp41-gp120 complex allowing gp41 to insert into the host-cell membrane. Gp41 now forms a direct bridge between the two membranes and thus constitutes a fusion peptide. The linkage between the virus and the cell are next re-enforced by a subsequent binding of CCR5 to a globular domain of gp120. In the last step, gp41 (fusion peptide) brings the two membrane so close that the lipid bilayers fuse with each other. The viral membrane now makes part of the cell plasma membrane and the capsid enters into the cytoplasm.
The human immunodeficiency virus (HIV) is a lentivirus (a subgroup of retroviruses that destroy the cells they infect) that causes HIV infection and, over time, the immunodeficiency syndrome (AIDS) because it infects some important cells of the immune system. The cells that are infected contain the CD4 receptor and a co-receptor (CCR5 or CXCR4). CD4 and CCR5, or CXCR4, are expressed on T helper lymphocytes which have a vital role in the adaptive immune reaction. In the end, the immune system doesn’t fulfill anymore its protective function and the HIV infected people become very sensitive to all type of infection.
HIV is around 120 nm in diameter (around 60 times smaller than a red blood cell) and roughly spherical. It is surrounded by an envelope of host-cell origin, which includes the glycoproteins gp120 and gp41, responsible for binding to and entering the cell. Like all retroviruses, it has reverse transcriptase and integrase and also some regulatory protein like tat (Trans-Activator of Transcription). The genetic information (RNA) is protected by a nucleocapsid so that it isn’t digested by nucleases.
The HIV genome has 9,7 kbp and it contains three major loci 5’gag-pol-env-3′ that code for capsid and matrix proteins, reverse transcriptase, integrase and HIV protease and glycoproteins (gp41, gp120), respectively. There are, of course, other genes that regulate the viral mechanisms. The gag gene gives the basic physical infrastructure of the virus, the pol provides the basic mechanism by which retroviruses reproduce, while the others help HIV to enter the host cell and enhance its reproduction.
Mutation and Resistance to the virus
The CCR5Δ32 mutation
CCR5-Δ32 is a variant of CCR5 gene (allele) that has appeared through a 32-base-pair deletion mutation that introduces a premature stop codon into the CCR5 receptor gene. As a result, the protein misses one interaction site with the viral gp120 protein. Carriers of the CCR5Δ32 gene and infected by HIV were found to be resistant: AIDS did not develop.
This allele is rare and has distinct geographic distribution indicating a single Northern origin followed by migration.
Exploiting CCR5 mutations in the treatment of HIV infection
Based on the above described findings, scientists have begun to develop ways of creating this mutation artificially. A novel genome engineering technique that goes under the name of CRISPR ( Clustered Regularly-Interspaced Short Palindromic Repeats) is a method to selectively modify genes. In the context of HIV, it can be used to artificially create the gene that codes for the CCR5-Δ32 protein. In order to achieve this, human bone marrow stem cells are extracted, their genome is modified with the CRISPR technique and the cells are then reinserted. This protocol has been tested with a positive outcome, the viral load is considerably reduced over time. For more information we suggest the following article: Allers K, Hütter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, Schneider T. Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood 2011;117:2791-9 or click on this link.