Retroviruses and Their Use As Tools for Cell Biology
Electron micrograph of HIV. Cone-shaped cores are sectioned in various orientations. Viral genomic RNA is located in the electron-dense wide end of core.
Retroviruses are infectious particles consisting of an RNA genome packaged in a protein capsid , surrounded by a lipid envelope . This lipid envelope contains polypeptide chains including receptor binding proteins which link to the membrane receptors of the host cell, initiating the process of infection. Retroviruses contain RNA as the hereditary material in place of the more common DNA. In addition to RNA, retrovirus particles also contain the enzyme reverse transcriptase (or RTase) , which causes synthesis of a complementary DNA molecule (cDNA) using virus RNA as a template.
Genetic organization Each retrovirus contains at least the following genetic sequence : LTR-GAG-POL-ENV-LTR This sequence is in the integrated provirus (DNA). LTR : Long Terminal Repeats ; in the viral DNA genes are bracketed by long terminal repeats (LTR), identical sequences that can be divided into three elements, including a promoter. These sequences are needed for successful transcription. GAG : G roup-specific A nti G en ; gene of the structural proteins (e.g. p24 and p17) POL : POL ymerase gene ; codes for protease, reverse transcriptase and integrase ENV : ENV elope gene ; codes for membrane proteins gp41 and gp120 Most Lentivirusses, including HIV-1, also have the following accessory genes : vif : viral infectivity factor vpr : viral protein R vpu : viral protein U rev : regulator of viral gene expression tat : trans-activator of transcription nef : negative effector These accessory genes are located downstream from POL. Some retroviruses (like RSV) carry an oncogene (like src).
In a addition to the nine proteins derived from GAG, POL and ENV, there are six other proteins made by HIV. Three of these are incorporated into the virus (Vif, Vpr and Nef) while the others are not found in the mature virus: Tat and Rev are regulatory proteins and Vpu indirectly assists in assembly. The genes that encode these proteins are known by three letter names that are derived as follows: TAT: T rans- A ctivator of T ranscription REV: Re gulator of V irion protein expression NEF: N e gative R egulatory F actor VIF: V irion I nfectivity F actor VPU: V iral P rotein U VPR: V iral P rotein R
The typical retrovirus genome consists of a single-stranded RNA of about 8500 nucleotides. The enzyme reverse transcriptase is a multifunctional enzyme that first makes a DNA copy of the viral RNA molecule. It then acts as a nuclease to remove the RNA, and then makes a second DNA strand, generating a double-stranded DNA copy of the RNA genome. The integration of this DNA into the host chromosome, catalyzed by a viral protein called integrase , is required for the synthesis of new viral RNA molecules by the host cell RNA polymerase. Retroviruses are examples of enveloped viruses , in which the protein shell is further enclosed by an outer lipid bilayer membrane. The envelope contains proteins that enable the virus to bind to cells, and that aid its entry into a cell. As indicated, the lipid membrane is acquired when the virus is released from the cell by a process of budding from the plasma membrane, taking some of the plasma membrane with it. The budding process is reversed when the virus reinfects a cell. Retrovirus Life Cycle
The retroviral life cycle begins in the nucleus of an infected cell. At this stage of the life cycle the retroviral genome is a DNA element integrated into and covalently attached to the DNA of the host cell. The genome of the virus is of approximately 8-12 kb of DNA (depending upon the retroviral species). Full-length genomic mRNA is made initiating at the beginning of the R (repeat) at the 5' LTR (Long Terminal Repeat). The free particle can infect new cells by binding to a cell surface receptor. The specificity of the virus-cell interaction is determined largely by the envelope protein(s) of the retrovirus. Infection leads to injection of the virus nucleoprotein core (consisting mostly of gag-derived proteins, full-length genomic RNA, and the reverse transcriptase protein). LTR LTR
Retroviruses contain viral RNA and several copies of reverse transcriptase (DNA polymerase). After infecting a cell, the reverse transcriptase is used to make the initial copies of viral DNA from viral RNA. Once a DNA strand has been synthesized, a complementary viral DNA strand is made. These double strand copies of viral DNA are inserted into the host-cell chromosome and host-cell RNA polymerase is used to make virus-related RNA. These RNA strands serve as templates for making new copies of the viral chromosomal RNA and serve also as mRNA. mRNA is translated into viral proteins that are used to make the virus envelope. New viral particles are assembled, bud from the plasma membrane, and are released. An example of this process is illustrated in the replication of the retrovirus, HIV (human immunodeficiency virus).
HIV infection begins with the interaction of the HIV glycoprotein gp120 with the CD4 molecule on the surface of the target cell. Following CD4 binding, a change in the HIV gp120/ gp 41 complex is induced by interaction of gp120 with the chemokine receptors CCR5 or CXCR4 . This change in confirmation exposes gp41 allowing it to initiate fusion of the membranes. The significant role of CCR5 in this process has been revealed upon the observation that individuals homozygous for mutations within CCR5 are resistant to infection by HIV-1. HIV Infection
As the virus fuses with the cell, internalization of the viral core with the associated RNA occurs. Partial uncoating of the viral core occurs to expose the viral RNA. Once in the cell cytoplasm, the conversion of the viral RNA into double-stranded DNA commences as the viral reverse transcriptase becomes active. HIV Infection
Reverse transcriptase synthesizes a double-stranded DNA copy of the single-stranded viral RNA generating a provirus . The viral DNA migrates to and enters the host cell nucleus (facilitated by HIV proteins vpr and MA ) and becomes integrated into the cell DNA with the help of the enzyme integrase . The provirus can then remain latent or be active, generating products for the generation of new virions. HIV Infection
Inside the nucleus, RNA polymerase II transcribes viral DNA into mRNA. The 9kb (genomic) mRNA is used for: 1. synthesis of the gag and gag-pol polyproteins 2. as the genetic material for the new virons formed. The 9kb mRNA can be spliced to yield 4kb and 2kb mRNAs. The 4kb viral mRNA is used to synthesize: 1. gp 120 2. gp 41 3. 3 regulating proteins - vif, vpr, and vpu. The 2kb viral mRNA is used to synthesize 3 regulatory proteins: tat, rev, and nef. HIV Infection
1) The viral mRNA leaves the nucleus. The translation of the viral mRNA results in the synthesis of three polyproteins : 2) ENV gp 160 - containing gp 120 and gp 41 3) GAG p55 - containing MA (matrix), CA (capsid), and NC (nucleocapsid protein) GAG-POL p 160 - containing MA (matrix), CA (capsid), PR (proteinase), (RT) reverse transcriptase, and INT (integrase) p55 and p160 are generated from the same mRNA strand by the process of ribosome frame shifting. The env gp160 proteins pass through the E.R. and Golgi apparatus to be processed into gp120 and gp41 HIV envelope proteins. During movement through the Golgi apparatus, glycosylation of gp120 occurs. RV Protein Processing
The gag and gag-pol polyproteins associate with the inner surface of the plasma membrane and interact with gp41 present in the plasma membrane. Some of the 9 kb viral RNA interacts with the nucleocapsid portion of p55. As p55 and p160 accumulate on the inner surface of the plasma membrane, they aggregate and commence assembly to form the viron. As assembly continues, the structure extrudes from the cell. Budding
As the virus buds from the cell, it acquires a lipid coat , carrying the gp120 and gp41 proteins. The virus is extruded into extra-cellular space in this immature state . During (or soon after) the budding of the new HIV particle from the host cell membrane, the viral proteinase in p160 becomes active, resulting in the cleavage of p160 and p55 into the various subunits and generating the mature form of HIV. This processing of p160 and p55 by the viral proteinase is essential for the generation of infectious virus. Budding and Maturation
Assembly of the virus occurs at the surface membrane of the cell. Viral Assembly gp41 p55 p160
The virus buds and the protease cuts itself free of the GAG-POL polyprotein . p55 gp120 p160 gp41 free protease
Further proteolytic cleavage occurs and the virus matures.
The necessity for CD4 antigen expression for entry of HIV into a human cell. HeLa and most other cells do not have CD4 antigen and are not infected. Cells transfected with CD4 gene can be infected with HIV. CD4 antigen gp120 CD4
Virus titer , CD4 cell number and anti-gp120 titer during the course of HIV infection. Time course of HIV infection
Wild-type retrovirus life cycle. 1) Viral particle with RNA genome binds to cell and is endocytosed. 2) Virus uncoats and RNA is reverse-transcribed into dsDNA. 3) dsDNA integrates into the infected cell’s genome irreversibly. 4) Viral genes are transcribed and translated into proteins. 5) New infectious viral particles assemble and bud from cell surface.
Use of retroviruses as expression vectors <ul><li>Viral genome is cloned on a DNA plasmid . </li></ul><ul><li>Cut out viral genes. Keep packaging sequence ( ps ). </li></ul><ul><li>3) Put in gene of interest, such as lacZ marker enzyme . </li></ul><ul><li>4) To make virus, cultured cells must be transfected with both the vector and helper DNA plasmids. </li></ul><ul><li>5) Helper plasmid expresses viral proteins necessary for viral particle formation, but does not contain a ps. </li></ul><ul><li>6) New vector particle cannot replicate itself (no viral genes). </li></ul>
Retroviral Vectors Retroviruses can be used to express two genes of interest in infected cells. pLZ10/LZ14 gag lacZ AATAAA pLZIS gag lacZ v- src IRES pBH1210 gag pol env pLZIS SH2 gag lacZ v- src SH2 IRES gag lacZ v- src SH3 IRES pLZIS SH3
lacZ only v-src v-src SH2 v-src SH3 Effects of v-src expression on developing brain cells Early developing brains were infected with retroviral vectors encoding only the lacZ marker gene , or lacZ along with different forms of the v-src tyrosine kinase. LacZ-expressing cells turn blue when incubated in X-gal.
Retroviral vector tumor cell marking 1) C6 rat glioma brain tumor cells were infected with a lacZ-containing retroviral vector. 2) Cells were injected into chick embryo brains and made tumors. 3) When tissue was incubated in X-gal, the tumor cells turned blue , so they are easy to find. White brain tissue, blue tumor cells.
Human U-87/lacZ Blue tumor cells can be seen in the wall of the brain.
Injecting Breast Cancer Cells for Metastasis Inject 50,000 cells in 5 l into E5 BV.
LacZ+ Breast Cancer Cells in E15 Brain Breast cancer cells have extravasated from the circulation and entered the brain.
Injection of retroviral vector into chick embryo brain
Normal “clones” of brain cells that came from single infected progenitor cells. The cells migrated radially in the developing brain along radial glia.
Chick embryo brains were infected with a mixture of 2 retroviral vectors. One encoded regular lacZ (right). The other (left) encoded lacZ that was fused to a nuclear localization signal sequence , so that the lacZ protein that was translated would enter and accumulate in the nucleus.
Migration of Clonal Cohorts Mechanism: Are integrins involved in radial migration? E3 E6 E7.5 E9 VZ VZ IZ MZ TP VZ
Antisense retroviral vectors Instead of expressing a second gene, like v-src, retroviral vectors can be used to mark cells (via lacZ) and shut down expression of a protein using antisense sequences . Essentially, this is a negative copy of a gene that is expressed by the retroviral vector, and it hybridizes to the cell’s mRNA to block it from being translated into protein. Therefore, you get specific attenuation of a gene. Here, antisense integrin 1 subunit sequences were used.
E 7 Clones lacZ RV 1) Antisense 1 integrin RV 2) Antisense 8 integrin RV 8 1 integrin is required for radial migration and cell survival. Antisense clones die E8- E12.
GFP-RV Can Be Used to Image and Recover Tumor Cells from Live Brain <ul><li>Colony assay to quantitate metastasis to brain after cell line modifications (e.g. AS-L1). </li></ul><ul><li>Serial injection/recoveries to select for subpopulation that targets brain. </li></ul>Breast cancer cells infected with GFP lentiviral vector . Cells recovered from a dissociated E15 embryo brain. Flat cells are normal brain cells. Live phase contrast/ fluorescence image.