Retroviral gene therapy

Introduction to retroviruses

Retroviral therapy is the use of retroviral vectors to provide remedy to disease via the genetic modification of a patient’s own cells. Retroviral vectors are themselves derived from natural retroviruses such as HIV. The name retrovirus refers to the unique ability of these viruses to convert viral RNA into DNA. A critical part of the viral life cycle is the integration of this viral DNA into the host cell’s genome, conferring a permanent genetic change to the cell. Therefore, retroviruses may be used as a vector for gene therapy, a method of treatment dealing specifically with the alteration of genes to achieve a therapeutic effect. Gene therapy techniques are divided into three main categories; viral(including retroviral gene therapy), nonviral, and physical. The use of retroviruses bears a significant advantage over these other forms of gene therapy. Nonviral and physical techniques are less efficient in transfection and, in the case of nonviral vectors, have a more limited expression. Viral techniques, however, are more efficient in transfection and better integrate viral genes into the target genome.

Introduction to retroviruses

Retroviruses belong to the family retroviridae and may be separated into three main subfamilies; oncoviruses, lentiviruses, and spuma viruses. While many retroviruses are benign, some are dangerously pathogenic. HIV for example, causes significant damage to the human immune system and is extremely deadly when left untreated. Retroviridae are some of the oldest viruses, emerging between 460 and 550 million years ago. One of the most important features of retroviruses is the permanent integration of viral genes into the DNA of their host. These genes are then inheritable by the offspring of the host. As a result, a significant portion of the vertebrae genome is derived from retroviral gene transfer. In fact, around 8% of the human genome consists of sequences incorporated by retroviral particles.

Integration of viral DNA is an important aspect of the retroviral life cycle. Retroviruses utilize a multitude of viral genes to accomplish their replication. All retroviral particles themselves consist of at least three genes, one which encodes for internal structural proteins(gag), one which encodes for the viral envelope(env), and one which encodes for a special type of polymerase called reverse transcriptase(pol). Reverse transcriptase is responsible for the conversion of viral RNA into DNA. All retroviruses contain prepackaged reverse transcriptases. Once a retrovirus has entered a host cell, the virus uncoats, releasing reverse transcriptases, RNA, and other viral molecules such as integrase into the cytoplasm. Immediately after uncoating, a reverse-transcription complex forms which initiates the conversion of viral RNA to DNA. As demonstrated in retroviruses like HIV, the viral RNA is first primed for transcription via the attachment of host t-RNA(Lysine t-RNA in HIV). After which, a reverse-transcriptase binds to the t-RNA bound viral RNA forming the reverse-transcription complex. The complex produces minus strand DNA which is further transcribed to form double stranded DNA. This double stranded is then integrated into the host DNA. To integrate, the integration complex must enter the nucleus. Therefore in some retroviruses, integration can only occur in dividing cells during the breakdown of the nuclear envelope. Others, however, are able to enter the nucleus through pores and are thus able to infect non dividing cells. The integration process begins with the removal of two nucleotides from each 3’end of the DNA exposing the terminal 3’hydroxyl group. After which the viral DNA is inserted into a random part of the host genome. Both processes are catalyzed by the viral enzyme integrase. Once integrated, viral DNA is referred to as the provirus.

The morphogenesis of a retroviruse from the provirus is divided into three stages; assembly, budding and maturation. All three stages rely on the gag polyprotein generated from the gag reading frame. In many retroviruses, it is believed that budding and maturation occur mainly near the plasma membrane of the host cell. Maturation, however, occurs after the retroviral particle buds off of the cell surface and is freed into the extracellular environment. Assembly begins with the provirus, which is transcribed by host mRNA machinery after integration. The first genes to be transcribed encode regulatory proteins including, Rev and Tat. While Tat upregulates transcription of the provirus, Rev enables the export of large unspliced viral mRNA transcripts. The largest of these transcripts is 9kb long and encodes for the gag protein. Once exported, the transcripts are translated by host ribosomes forming viral proteins. Once formed the gag protein mediates the recruitment of the viral genomic RNA. A region of the protein termed the nucleocapsid domain selects for two copies of the viral genome, binding them using a packaging signal located near the 5’ end of the transcript. After which, the Gag protein and the attached RNA move to the plasma membrane to begin assembly. Once at the plasma membrane, Gag acts as a structural protein, coordinating the construction of the immature viral molecule from a mix of host protein, viral protein, and lipids as well as gag itself. In HIV, Gag protein makes up around 50% of the entire virion while host proteins as well as other viral proteins make up an estimated 20% of the HIV particle. The gag protein consists of four domains, the previously discussed nucleocapsid region, the MA region, the CA region, and the C terminus region p6. During assembly, the MA region is primarily responsible for binding to the plasma membrane while the CA region mediates assembly and promotes the construction of the viral shell. Once both processes are complete, the virus moves on to the budding stage. To initiate budding, retroviruses hijack the host’s ESCRT pathway which is conventionally used to create multivesicular endosomes. As budding occurs, the virion crosses the cell’s plasma membrane using it to form its own lipid envelope. The gag protein plays a major role in budding by recruiting ESCRT proteins to the release site. This step helps to mediate membrane fission and release. During recruitment, the ESCRT proteins CHMP1, CHMP2, and CHMP4 facilitate closure of the viral membrane’s neck. These ESCRT establish a spiraling filament at the neck constricting both the cell and viral membrane. After, membrane fission is directed by the recruitment of VPS4 ATPases which initiate the breakdown of ESCRT assemblies thereby catalyzing release of the viral particle. After the virion releases from the membrane, it enters the final stage of replication, maturation. Maturation involves the structural rearrangement of the virion via the cleavage of the gag protein via viral protease at a number of sites. The result of this cleavage is the production of multiple processed proteins which then rearrange dramatically. Part of this rearrangement involves the encapsidation of the genomic RNA into a viral core which can be injected into a new host cell. After rearrangement is completed, the virion has matured and is capable of infecting other host cells.