NPTEL – Biotechnology – Gene Therapy Module 3 Viral vectors Lecture 15 Lentivirus Lentiviruses are enveloped RNA viruses found in most of the vertebrates. The most common Lentiviruses used in gene therapy experiments are retroviruses. The virion contains two copies of RNA genome, which are covered by a cone shaped core. The viral genome contains three genes that code for different viral proteins. The gag gene encodes for capsid, matrix, and nucleocapsid protein. The pol gene is a viral polymerase which comprises of proteases, reverse transcriptase and integrase. The env gene encodes for glycosylated envelope protein which mediates the virus entry into the host cell receptor. The viral RNA genome is flanked by long terminal repeats (LTR) sequences which are responsible for packaging of viral RNA genome. Most of the retroviruses lead to persistent infection by integrating to the host cell genome. Lentiviruses also contain two additional proteins tatand rev which take part in transactivation of viral transcription and nuclear export of viral RNA, respectively. Following the infection virion binds to the host cell receptor and viral genome enters the cell by fusion. Viral RNA is converted into double stranded DNA with the help of enzyme reverse transcriptase. The double stranded DNA then migrates to the nucleus and integrates to the host cell genome by the help of enzyme integrase. This stage of the virus is known as Provirus. The proviral DNA forms new viral genome by using cellular transcription factors. The immature viral proteins are processed by viral proteases, and assemble along with RNA genome to form an infectious virion, which then buds out from the host cell membrane. Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 55 NPTEL – Biotechnology – Gene Therapy Most of the work based on the lentiviral vector focuses on modifying the human immune deficiency virus type 1 (HIV-1). HIV-1 encodes six accessory proteins namely tat, rev, vif, vpr, nef, and vpu in addition to an essential gag, pol, and env proteins (Figure 15.1). Table 15.1 Different genes of lentivirus and their functions: Gene Name Function gag Group specific antigen Nucleocapsid core protein pol Polymerase Encode reverse transcriptase, integrase, protease vif Viral infectivity fector Virus infectivity vpr Viral protein R Nuclear targeting vpu Viral protein unique Help in virus budding env Envelope Codes for surface coat protein tat Transactivator of Virus gene expression transcription rev Regulator of expression of Structural gene expression virion proteins nef Negative regulatory factor Virus infectivity Figure 15.1 Schematic diagram of lentivirus: Joint initiative of IITs and IISc – Funded by MHRD Page 2 of 55 NPTEL – Biotechnology – Gene Therapy Gene therapy vectors based on lentiviruses lack all the viral sequences except the LTRs, rev responsive element (RRE), and cis-acting elements. Usually viral rev proteins are added in transin order to facilitate the trafficking of viral RNA genome in the nucleus following efficient binding with RRE. The production of vector RNA is either directed by LTRs or by tissue specific promoter. Generally vectors are designed in such a way so as to utilize tissue specific promoter (CMV) and LTR hybrid promoter (CMV/LTR). The use of CMV/LTR hybrid promoter allows abundant vector RNA production independent of transactivation by tat protein. HIV accessory proteins, vif, vpr, nef, and vpucan be deleted during the lentiviral production. Sometimes polypurine tract present in the HIV genome can be included as a cis- active element during the viral production in order to enhance the nuclear trafficking. The HIV-1 infects only the cells that contain CD4 receptor and co-receptors such as CXCR4 and CCR5.The attachment of HIV to the receptor and co-receptors is largely mediated by viral glycoproteins. This specificity restricts the host range for HIV-1 infection. Scientists worldwide are trying to broaden the host range by various molecular biology techniques. One such technique is to generate a pseudo type HIV virus that contains vesicular stomatitis glycoprotein along with env protein, which is having broad tropism. Figure 15.2 Generation of lentiviral vector: Diagram depicts a HIV based vector system where viral essential genes have been replaced by the transgene. Cytomegalovirus promoter is used to transcribe the transgene as well as gag, RRE, and cPPT. RRE acts a cis-acting sequence essential for nuclear export of viral RNA while polypurine tract (cPPT) helps in the import of proviral DNA. The deletion in U3 region in the genome makes it inaccessible to use LTR for transcription. The packaging system consists of gag/pol, VSV-G, and rev expressing constructs. Presence of RRE along with gag/pol helps in nuclear export of the RNA by transcribing rev protein.Lentiviral vectors are produced by transfection of vector construct along with the packaging constructs in producer cells. Vector RNA genomes are packaged by precursor proteins atthe cellular membrane. The mature particles bud through the cellular membrane,containing the envelope derived from VSV-G glycoproteins. Joint initiative of IITs and IISc – Funded by MHRD Page 3 of 55 NPTEL – Biotechnology – Gene Therapy Joint initiative of IITs and IISc – Funded by MHRD Page 4 of 55 NPTEL – Biotechnology – Gene Therapy Lecture 16 Recombinant simian virus 40 Simian virus 40 (SV40)belongs to polyoma viruses group. The virus was first isolated as a contaminant from a monkey kidney culture used for the production of poliovirus vaccine. Its genome is very small and can be easily modified for gene therapy purpose. Its genome has ds circular DNA wrapped up as nucleosome with the help of histone proteins (H1,H2 (A and B), H3, H4). It is roughly 40-45 nm in diameter. The chromosome is 5000bp long.The genome of SV 40 is like a minichromosome. The genome of SV40 consists of early proteins, late proteins and regulatory proteins. Early proteins are non structural while late proteins are structural proteins. Regulatory region consist of promoter, enhancer and origin of replication.The virions are made up of three capsid proteins namely VP1, VP2, and VP3. MHC class I molecules act as receptors for SV 40 virus. Following attachment to the cell surface by VP1 virus gets internalized by endocytosis.The viral genome replication takes place inside the nucleus (Figure 1). The host RNA polymerase II helps in the transcription of early gene products. The early proteins produced by the virus once it enters the cell consist of T antigen, t antigen and middle T antigen. The large T antigen migrates to the cytoplasm while majority of it gets back to the nucleus duringvirus formation before release. The capsid proteins are produced later which then forms viable virus and comes out of the cell.The early protein coding genes can be replaced by the gene of interest.The mutation in p53 promoter which leads to cancerous condition was discovered in the SV 40 virus. Joint initiative of IITs and IISc – Funded by MHRD Page 5 of 55 NPTEL – Biotechnology – Gene Therapy Figure 16.1 Replication of SV40 in the host cell: 16.1 SV40 as a vector Gene therapy using recombinant SV40 is highly suitable means to transfer the gene in a number of conditions. The wild type SV40 virus encodes two early genes, a large T antigen and a small t antigen. The large T antigens are important for virus replication and synthesis of viral late proteins. The genome of SV40 can be modified in order to carry foreign gene by deleting large T antigen (approximately 2.5 kb). The most alarming situation in a wild type SV40 is regarding the ability of large T antigen to bind with tumor suppressor genes such as p53 and retinoblastoma (pRb) protein. The binding of large T antigen may lead to cancer because of inactive p53 and pRb proteins. To avoid this complication all SV40 recombinant viruses lack large T antigen. The late proteins of SV40 are synthesized from the opposite strand. Out of four structural proteins VP1 is the most abundant one. The minimum viral specific sequence required to assemble the virion consists of origin of replication and encapsidation sequences.The origin of replication and encapsidation sequences overlaps with the early promoter region in the genome. The transcription of the genome and the transgene is governed by pol III promoter. The transcription of the transgene is terminated by the incorporation of a polyadenylation signal downstream to the gene of interest. Most of the vectors available for the expression of transgene requires SV40 early and late polyadenylation signal. Joint initiative of IITs and IISc – Funded by MHRD Page 6 of 55 NPTEL – Biotechnology – Gene Therapy Recombinant SV40 viral vectors are made in some plasmid background such as pGEMT plasmid vector. The permissible cells are transfected with the plasmid along with the support plasmid needed to package the viral genome. The recombinant virus containing the transgene is then recovered by the lysis of transfected cell. The recombinant virus is then further amplified in the next round of infection in the cell lines. This approach is used to package the transgene of about 5.5 kb without affecting the productivity. 16.2 Features of a SV40 based vector Some of the specific features of an SV40 vector are as follows Capacity of the transgene is limited to 5 Kb. Both resting as well as dividing cells are equally transduced by SV40 based vectors. High level of transduction efficiency. Long lasting transgene expression. It has been reported that the transgene expression once established will remain lifelong. Neutralizing antibody against recombinant SV40 has not been reported yet. Therefore it is easy to administer multiple injection of recombinant SV40 vector without neutralizing its effect. SV40 readily integrates with the host cell genome both in dividing and non dividing cells.