As a member of the retroviridae family, Lentivirus has become an increasingly important tool for gene delivery to many cell types. Lentivirus has also been applied in clinical trials for gene therapy to treat genetic disorders.  Like other retrovirus,  lentivirus is characterized by the ability to retro-transcribe its RNA genome into a DNA copy, which is then stably integrated into the target cell’s genome. Retrovirus can be divided into the simple (e.g., murine leukemia virus) or complex (e.g. lentivirus) group. The main difference is the presence of a number of accessory and regulatory genes in complex (lentivirus), but not simple, retrovirus. The viral particles or virion of both groups contain two copies of positive-stranded RNA associated with an viral reverse transcriptase (RT) within a core, which also contains structural and enzymatic proteins, such as the nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR). The inner core is surrounded by an outer protein layer, comprised of the matrix (MA) protein, which is in turn encompassed by the envelope (ENV) protein-studded, host cell membrane-derived envelope (see the retroviridae structure and discussion below).

Lentivirus Genome

Like simple retrovirus, lentivirus contains two copies of linear, single-stranded RNA of 7-12 kb in length that encode the gag, pol, and env genes. Gag encodes a polyprotein that is translated from one mRNA which is then cleaved by the viral protease (PR) into the MA, CA, and NC proteins (see the diagram below). The Env gene also encodes a polyprotein which is in turn cleaved by a cellular protease into the surface (SU) envelope glycoprotein, gp120 and the transmembrane glycoprotein, gp41. While gp120 interacts with the cellular receptor and co-receptor, gp41 anchors the gp120 complex in the viral membrane and catalyzes membrane fusion with the target cell during the entry.

Pol is expressed as a Gag-Pol polyprotein, producing the enzymatic proteins RT, PR, and IN, which are associated with the viral genome within the core. The RT protein possesses three distinct activities: (a) RNA-dependent DNA polymerase activity, responsible for transcribing the two RNA genomes into a single DNA; (b) RNAse H activity; and (c) DNA-dependent DNA polymerase activity. PR cleaves the Gag and Gag-Pol polyproteins, resulting in the maturation and production of fully infectious virions. IN is responsible for the integration of viral DNA into the host genome. Once integrated, the viral genome is contiguous with the host cell chromosome and is also referred to as a provirus (see the provirus genome structure below). 

The lentivirus genome also encodes a set of accessory genes whose products are involved in the regulation of transcription, RNA transport, gene expression, and virion assembly. They include Rev and Tat as well as the accessory proteins, Vpu, Vif, Vpr, and Nef. Rev is an RNA-binding protein that promotes late phase gene expression. It is also important for the transport of mRNAs of viral genome, from the nucleus to the cytoplasm. Tat is an RNA-binding protein that enhances transcription. The Nef protein inhibits T-cell activation. Vpu enhances the release of the virus from the cell surface to the cytoplasm during entry. The Vif protein is necessary for replication of lentivirus due to its ability to down regulate the host’s antiviral response.

Provirus Genome

The integrated provirus genome has 5’ and 3’ long terminal repeats (LTR) which each consist of three regions: (1) the U3 region, which functions as a promoter and contains transcriptional enhancer elements and a TATA box; (2) the R region, which is where transcription begins; and (3) the U5 region, which is involved in reverse transcription and carries a tRNA primer-binding site. Other important elements of the provirus are the packaging signal (ψ, psi) and the polypurine tract (ppt), which serves as the site of initiation of positive-strand DNA synthesis during reverse transcription.

Lentivirus Replication

The replication cycles of lentivirus begins when the envelope glycoprotein gp120 binds the cellular receptor, resulting in a conformational change in ENV and the fusion of the virion envelope and cellular membrane, leading to the release of the viral core into the cytoplasm of target cell. Viral tropism is determined by recognition of specific cellular receptors by the viral envelope glycoprotein, gp120. For example, HIV recognizes CD4 on helper T-lymphocytes followed by interaction with the co-receptor, typically CXCR4 or CCR5.

While still in the viral core, the RNA genomes are reverse transcribed by RT into double-stranded DNA. During lentivirus infection, the viral dsDNA is transported into the nucleus (unlike the simple retrovirus, which can only be transported into the nucleus during cell division). Therefore lentivirus is capable of integrating into the genome of both dividing and non-dividing cells, whereas the simple retrovirus can only transduce dividing cells. After the accumulation of newly synthesized viral proteins and viral genomic RNA, these components are packaged and bud from the cell, after acquiring a host cell-derived membrane (see the diagram below).

Lentivirus Applications and Hurdles

Primary cells such as stem cells can be genetically modified using lentivirus and then introduced into animals. Lentivirus can also replace the injection of DNA into fertilized oocytes as a way to produce transgenic animals. Lentivirus also shows promise in medical applications. However safety concerns are major hurdles, e.g., the generation of replication-competent virus and potential oncogenesis & genotoxicity. There are also challenges to produce clinical grade virus with the required purity and yield. Progress continues to be made in rendering lentivirus safer and more useful in clinic. For example, integration-defective lentiviral systems, which contain a mutated IN lacking enzymatic activity, have been developed. They can produce double stranded episomal DNA circles in the host cell nucleus without stable integration, thereby avoiding the potential oncogenesis due to the random integration and insertion.

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Many virus-based gene delivery systems have been developed to exploit viral transduction for inheritable or sustainable expression of a transgene in model organisms. The following table provides a comparison of key features of 4 major virus-based delivery systems, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. Each system has advantages and disadvantages.  For examples,  all 4 systems show broad tropism, capable of transducing a wdie range of host, tissue and cell types.  Adenovirus has high transduction efficicency and is easy to amplify to high titers (>10¹² TU/ml).  Adenovirus does not integrate into the host genome, but it induces strong inflammatory response when administered in vivo.  In contrast, AAV is almost non-pathogenic and triggers little or no immune response.  However AAV has the smallest packaging capacity (4-5 kb only) among all 4 systems. Retrovirus can only transduce dividing cells, while other 3 systems can infect both dividing and non-dividing cells.  A unique feature of retroviral and lentiviral systems is that they produce replication defective viral particles. This allows for the delivery of the desired transgene without continued viral replication in the target cells, thus, eliminating the dangers associated with the use of a live pathogen.  However the risk of genome integration-induced mutagenesis and potential activation of oncogenesis limits the clincal applications of retroviral and lentiviral systems.

 Comparison of Different Viral Gene Delivery Systems

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