A lentivirus is a type of retrovirus, a class of viruses with a genome composed of ribonucleic acid (RNA) rather than the deoxyribonucleic acid (DNA) found in most organisms. This RNA-based genome contains all the instructions the virus needs to replicate within a host. The complexity of the lentiviral genome allows it to perform sophisticated actions, setting it apart from simpler retroviruses. A lentivirus particle consists of a spherical envelope containing its genetic material: two identical single strands of RNA. This genetic blueprint dictates the virus’s entire life cycle and is key to understanding how it can be adapted for other purposes.
Genomic Structure and Essential Genes
The lentiviral genome is about 9,700 nucleotide bases long and organized into several genes. The primary structural genes, common to all retroviruses, are `gag`, `pol`, and `env`. The `gag` gene codes for the inner structural proteins, such as the matrix and capsid, which are responsible for the physical assembly of new viral particles.
The `pol` gene produces enzymes for the replication cycle: reverse transcriptase converts viral RNA to DNA, integrase inserts this DNA into the host genome, and protease processes viral proteins. The `env` gene codes for the envelope glycoproteins on the virus’s outer surface. These proteins allow the virus to recognize and bind to receptors on a host cell, facilitating entry.
Flanking the protein-coding regions are two non-coding sequences known as Long Terminal Repeats (LTRs), located at each end of the RNA. The LTRs contain signals for reverse transcription and are recognized by the integrase enzyme for insertion into the host chromosome. The 5′ LTR also acts as a promoter, initiating the transcription of viral genes using the host cell’s machinery.
Lentiviruses also possess regulatory and accessory genes that increase their efficiency. The two primary regulatory genes are `tat` and `rev`. The Tat protein enhances the rate of viral gene transcription, while the Rev protein controls the splicing and export of viral RNA from the nucleus. Other accessory genes help the virus overcome host defenses and optimize replication.
The Replication Cycle and Genomic Integration
The lentiviral life cycle begins when its envelope proteins mediate attachment and fusion with a host cell membrane. The viral core is then released into the cell’s cytoplasm and uncoats, exposing its RNA genome and enzymes. The reverse transcriptase enzyme begins the process of reverse transcription, reading the RNA genome to synthesize a complementary strand of DNA.
This initial RNA-DNA hybrid serves as a template to create a double-stranded DNA copy of the viral genome, reversing the standard flow of genetic information. The resulting viral DNA, as part of a pre-integration complex, is then transported into the host cell’s nucleus.
Lentiviruses can infect non-dividing cells, like neurons, because their pre-integration complex can pass through the intact nuclear envelope. Once inside the nucleus, the integrase enzyme permanently inserts the viral DNA into the host’s chromosomal DNA. This integrated viral DNA is known as a provirus.
Once integrated, the provirus uses the cell’s machinery to be transcribed back into RNA. Some of these new RNA molecules become genomes for the next generation of viruses, while others are translated into viral proteins. These new proteins and RNA genomes assemble at the cell membrane, forming immature viral particles that bud off to infect other cells.
Engineering Lentiviral Genomes for Therapeutic Use
The ability of lentiviruses to integrate their genome into host cells makes them a useful tool for gene therapy. Scientists engineer the lentiviral genome into a delivery vehicle, or vector, by removing the genes associated with replication. The `gag`, `pol`, and `env` genes are deleted from the viral genome that will be delivered to the patient.
A therapeutic transgene is inserted into the modified genome in place of the removed viral genes. This transgene provides a therapeutic benefit, such as a functional copy of a faulty gene. The engineered genome retains non-coding elements, including the LTRs and a packaging signal (Psi), which allows the viral RNA to be packaged into new particles.
To produce vector particles, the engineered genome is introduced into specialized “producer” cells. These cells are also supplied with the missing viral genes (`gag`, `pol`, and `env`) on separate DNA molecules called helper plasmids. The producer cells use these plasmids to manufacture the viral proteins, which then assemble around the therapeutic vector genome to create a complete, infectious particle.
The resulting lentiviral vectors can infect target cells and deliver their therapeutic payload. Because the vector lacks the genes for `gag`, `pol`, or `env`, it is replication-defective. Once it delivers its therapeutic gene, it cannot produce new viral particles, which prevents the vector from spreading after administration.
Safety Mechanisms and Vector Design
Advanced designs incorporate multiple layers of protection to enhance the safety of lentiviral vectors. One such design is the self-inactivating (SIN) vector, where a portion of the 3′ LTR is deleted. During reverse transcription, this modified LTR is copied to the 5′ position of the integrated provirus. This deletion disables the LTR’s promoter activity, effectively “switching it off” once inside the host genome.
This self-inactivation mechanism reduces the risk of insertional mutagenesis. This event occurs if the vector integrates near a proto-oncogene (a gene with the potential to cause cancer) and the LTR’s promoter activity activates it. By silencing the LTR, SIN vectors minimize the chance of activating nearby host genes, making them safer for clinical applications.
A split-genome packaging system adds another layer of security. Modern third-generation systems split the viral genes across separate helper plasmids, such as one for `gag` and `pol`, another for `rev`, and a third for `env`. This separation of components makes it highly improbable for the plasmids to recombine and create a replication-competent virus.
Scientists can also control which cells a vector infects through a process called pseudotyping. This involves replacing the native `env` gene on a helper plasmid with an envelope gene from a different virus, like Vesicular Stomatitis Virus G-protein (VSV-G). Using VSV-G allows the vector to infect a wide variety of cell types. Conversely, other envelope proteins can be used to target the vector to a specific tissue, increasing therapeutic precision.