Reverse transcription is the process by which the viral RNA genome is converted into a double strand of DNA. HIV-1 reverse transcription is affected by a number of viral proteins including one called Tat. Genetic analysis showed that Tat’s function in reverse transcription was distinct from its role in HIV-1 gene expression where it can activate HIV as much as a thousand-fold. The mechanism by which Tat enhances reverse transcription remains unknown, but the observation that Tat-deficient virus can be trans-complemented for reverse transcription in a virus producer cell but not in the target cells indicates that Tat has an early effect on the virion or is itself a virion protein. Our experiments have shown that key mutations in Tat can effectively block virus replication by inhibiting reverse transcription.
Tat is a virion protein but exactly how this predominantly nuclear protein traffics to the plasma membrane is unknown. A current grant awarded by the National Health and Medical Research Council aims to investigate Tat-enhanced reverse transcription, mechanisms of Tat trafficing, and methods to block Tat function.
Viral RNA regulates reverse transcription
We have investigated a RNA stem-loop structure called the transactivation response element (TAR), which is best known for its role in supporting high levels of HIV-1 gene expression. Viruses with mutations in TAR show a greatly reduced ability to support reverse transcription. How TAR functions in reverse transcription is unknown, but at least two reasonable mechanisms can be proposed. TAR RNA may directly interact with viral protein(s) or with other RNA sequences in order to stabilise a reverse transcription complex. Alternatively, TAR may recruit to the virion a factor(s) which can enhance the reverse transcription reaction.
Cellular factors and HIV reverse transcription
The studies described above have primarily focused on the initiation of reverse transcription. Our data indicate that a post-entry event or cell factor may be involved in efficient HIV-1 reverse transcription in vivo. We seek to determine factors which enable late HIV-1 DNA synthesis with the long term aim to define new drug targets. We have demonstrated an in vitro system that can be used to identify cellular factors required by HIV-1 for efficient reverse transcription. A current grant awarded by the National Health and Medical Research Council aims to identify the key factors required for virus replication after entry into a cell.
Protein Arginine Methylation
Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. Recently, it has been shown that inhibiting protein methylation can modulate infectivity in some viruses. Our data have shown that protein methylation is required for optimal HIV-1 infectivity. When protein methylation was inhibited in cells producing HIV-1 using adenosine periodate, the resultant virus was affected in terms of virion size, virion composition and infectivity. We are investigating which viral proteins are methylated and to identify the relevant protein methyltransferase activity.
Outside of a human cell, HIV exists as roughly spherical particles (sometimes called virions). The surface of each particle is studded with lots of little spikes.
An HIV particle is around 100-150 billionths of a metre in diameter. That’s about the same as:
- 0.1 microns
- 4 millionths of an inch
- one twentieth of the length of an E. coli bacterium
- one seventieth of the diameter of a human CD4+ white blood cell.
Unlike most bacteria, HIV particles are much too small to be seen through an ordinary microscope. However they can be seen clearly with an electron microscope.
HIV particles surround themselves with a coat of fatty material known as the viral envelope (or membrane). Projecting from this are around 72 little spikes, which are formed from the proteins gp120 and gp41. Just below the viral envelope is a layer called the matrix, which is made from the protein p17.
The proteins gp120 and gp41 together make up the spikes
that project from HIV particles, while p17 forms the matrix
and p24 forms the core.
The viral core (or capsid) is usually bullet-shaped and is made from the protein p24. Inside the core are three enzymes required for HIV replication called reverse transcriptase, integrase and protease. Also held within the core is HIV’s genetic material, which consists of two identical strands of RNA.
HIV belongs to a special class of viruses called retroviruses. Within this class, HIV is placed in the subgroup of lentiviruses. Other lentiviruses include SIV, FIV, Visna and CAEV, which cause diseases in monkeys, cats, sheep and goats. Almost all organisms, including most viruses, store their genetic material on long strands of DNA. Retroviruses are the exception because their genes are composed of RNA (Ribonucleic Acid).
RNA has a very similar structure to DNA. However, small differences between the two molecules mean that HIV’s replication process is a bit more complicated than that of most other viruses.
HIV has just nine genes (compared to more than 500 genes in a bacterium, and around 20,000-25,000 in a human). Three of the HIV genes, called gag, pol and env, contain information needed to make structural proteins for new virus particles. The other six genes, known as tat, rev, nef, vif, vpr and vpu, code for proteins that control the ability of HIV to infect a cell, produce new copies of virus, or cause disease.
At either end of each strand of RNA is a sequence called the long terminal repeat, which helps to control HIV replication.
HIV life cycle
HIV can only replicate (make new copies of itself) inside human cells. The process typically begins when a virus particle bumps into a cell that carries on its surface a special protein called CD4. The spikes on the surface of the virus particle stick to the CD4 and allow the viral envelope to fuse with the cell membrane. The contents of the HIV particle are then released into the cell, leaving the envelope behind.
Reverse Transcription and Integration
Once inside the cell, the HIV enzyme reverse transcriptase converts the viral RNA into DNA, which is compatible with human genetic material. This DNA is transported to the cell’s nucleus, where it is spliced into the human DNA by the HIV enzyme integrase. Once integrated, the HIV DNA is known as provirus.
Transcription and Translation
This electron microscope photo shows
newly formed HIV particles budding
from a human cell.
HIV provirus may lie dormant within a cell for a long time. But when the cell becomes activated, it treats HIV genes in much the same way as human genes. First it converts them into messenger RNA (using human enzymes). Then the messenger RNA is transported outside the nucleus, and is used as a blueprint for producing new HIV proteins and enzymes.
Assembly, Budding and Maturation
Among the strands of messenger RNA produced by the cell are complete copies of HIV genetic material. These gather together with newly made HIV proteins and enzymes to form new viral particles, which are then released from the cell. The enzyme protease plays a vital role at this stage of the HIV life cycle by chopping up long strands of protein into smaller pieces, which are used to construct mature viral cores.
The newly matured HIV particles are ready to infect another cell and begin the replication process all over again. In this way the virus quickly spreads through the human body. And once a person is infected, they can pass HIV on to others in their bodily fluids.
Fusion of HIV with a target cell
- The gp120 molecule of HIV first interacts with the CD4 antigen of the target cell.
- Heparan sulfate proteoglycan stabilizes the interaction of the gp120 with CD4 antigen
- This interaction induces a conformational change in the gp120 exposing sites that interact with the chemokine receptor (CCR5 or CXCR4)
- This further stablizes the interaction and the virus fusion protein (gp41) is uncovered and also undergoes a conformational change
- Gp41 inserts into the membrane of the host cell to initiate the fusion of the two bilayers
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