Team:Freiburg/Content/Project/The viral vector

From 2014.igem.org

(Difference between revisions)
 
(47 intermediate revisions not shown)
Line 5: Line 5:
</head>
</head>
<body>
<body>
-
<h1>Retroviral introduction</h1>
+
 
-
<section>
+
<div class="row category-row">
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: left">
 +
<div style="position: relative; float: right; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">Go back to The Receptor</div>
 +
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
 +
</div>
 +
</div>
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: right">
 +
<div style="position: relative; float: left; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Project/Vision">Read more about Mammalian Systems for iGEM</div>
 +
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
 +
</div>
 +
</div>
 +
</div>
 +
 
 +
<section id="Project-TheViralVector">
 +
<h1>The Viral Vector</h1>
 +
 
 +
<h2 id="Project-TheViralVector-Introduction">Retroviral Introduction</h2>
 +
 
<div class = "row category-row">
<div class = "row category-row">
<div class = "col-sm-6">
<div class = "col-sm-6">
-
<p>Retroviruses are ubiquitous viruses, found in fish, avian and mammals. The diameter of the retroviral virion is approximately 80 &ndash; 100 nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located.</p>
+
<p>Retroviruses are enveloped viruses that are found in fish, avian and mammals. The diameter of a retroviral virion is approximately 80 – 100&nbsp;nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located (Fig. 2). They are positive-sense and approximately 7 – 11 kb long [5]. The retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Moloney Murine Leukemia Virus (MuLV), which is the origin of the viral vector we are using for our project.</p>
</div>
</div>
<div class = "col-sm-6">
<div class = "col-sm-6">
-
<p> They are positive-sense and approximately 7 &ndash; 11 kB long (5)</p>
+
    <figure style="max-width: 400px">
-
<p>Generally the retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Molony Murine Leukemia Virus, which is the origin of the viral vector we are actually using.</p>
+
        <a href="https://static.igem.org/mediawiki/2014/1/13/Freiburg2014_ViralVector_Integration.png">
 +
                <img src="https://static.igem.org/mediawiki/2014/1/13/Freiburg2014_ViralVector_Integration.png">
 +
        </a>
 +
        <figcaption>
 +
<p class="header">Fig.1: Steps in viral transduction.</p>
 +
        </figcaption>
 +
    </figure>
</div>
</div>
</div>
</div>
</section>
</section>
-
<h1>Retroviral tropism and infection mechanism</h1>
 
<section>
<section>
 +
<h2 id="Project-TheViralVector-TropismAndInfectionMechanism">Retroviral Tropism And Infection Mechanism</h2>
<div class = "row category-row">
<div class = "row category-row">
<div class = "col-sm-6">
<div class = "col-sm-6">
-
<p>Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect (ref). The Molony Murine Leukemia Virus (MMLV) is an ecotropic gammaretrovirus; it only infects rodent cells (mice and rat)(ref). This tropism of the virus is granted by its high specificity towards the mCAT1 receptor (ref). Once attached to the receptor the MMLV outer membrane fuses with the host cells membrane and the single stranded viral RNA genome is transferred into the cytoplasm. Here it is reverse transcribed into ds DNA by the viral reverse transcriptase. </p>
+
<p>Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect [1]. The Moloney Murine Leukemia Virus (MuLV) is an ecotropic gamma-retrovirus; it only infects rodent cells (mice and rat). This tropism of the virus is granted by its high specificity towards the mCAT-1 receptor [7]. Once attached to the receptor the MuLV enters the target cell and the single stranded viral RNA is transferred into the cytoplasm.</p>
</div>
</div>
<div class = "col-sm-6">
<div class = "col-sm-6">
</div>
</div>
-
<p>After the conversion the dsDNA is further processed by the viral Integrase protein. Integrase is recessing the 3&rsquo; ends of the ds DNA by 2 bp (1). When the cell is going through mitosis the ds DNA can reach the host cells genome due to the breakdown of the nuclear membrane, there the target sequence is cleaved and the 5&rsquo; ends are recessed. After integration the dsDNA is called provirus. The provirus starts expression of its genes.</p>
+
<p> Within the cell a complementary strand of DNA is created by the viral reverse transcriptase, which is then replicated to form a double strand DNA (dsDNA). During mitosis the dsDNA can enter the nucleus due to the breakdown of the nuclear membrane. Once in the nucleus the viral DNA is further processed by the viral Integrase that recesses the the 3&rsquo; ends of the DNA by 2 bp. Double stranded viral DNA is then integrated at random into the host genome and, termed as provirus, it starts to express viral enzymes and proteins.   </p>
</div>
</div>
</section>
</section>
-
<h1>The retoviral genome</h1>
+
 
<section>
<section>
 +
<h2 id="Project-TheViralVector-Genome">The Retoviral Genome</h2>
<div class = "row category-row">
<div class = "row category-row">
<div class = "col-sm-6">
<div class = "col-sm-6">
-
<p>MMLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. Besides this trans-gene elements there are also regulatory cis-gene elements. The trans-gene elements are flanked by long terminal repeats (LTR). The 5&rsquo; LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3&rsquo; LTR is needed for polyadenylation of the proviral mRNA. The gag (group antigenes) gene is precursor polyprotein which is further processed after translation. After procession it forms the major proteins building up the core particle, RNA binding proteins and the nucleoprotein core particle.</p>
+
<p>The MuLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. These so called trans-gene elements are flanked by long terminal repeats (LTR). The 5 &rsquo; LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3&rsquo; LTR is needed for polyadenylation of the proviral mRNA. The gag (group specific antigen) genes are precursor polyproteins that form the major components building up the core particle, RNA binding proteins and the nucleoprotein core particle. The pol genes encode for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. The retroviral psi-packaging sequence is a cis-acting RNA element which allows the transcribed viral RNA to be incorporated into the assembly of the new virus [3]. After proper packaging the virus is leaving the cell and can start the next round of infection.</p>
-
     <figure>
+
</div>
 +
<div class = "col-sm-6">
 +
     <figure style="max-width: 400px">
         <a href="https://static.igem.org/mediawiki/2014/4/43/2014Freiburg_Virus_Kopie.png">
         <a href="https://static.igem.org/mediawiki/2014/4/43/2014Freiburg_Virus_Kopie.png">
                 <img src="https://static.igem.org/mediawiki/2014/4/43/2014Freiburg_Virus_Kopie.png">
                 <img src="https://static.igem.org/mediawiki/2014/4/43/2014Freiburg_Virus_Kopie.png">
         </a>
         </a>
         <figcaption>
         <figcaption>
-
<p class="header">Fig.1: Schematic retrovirus.</p>
+
<p class="header">Fig.2: Schematic retrovirus.</p>
         </figcaption>
         </figcaption>
     </figure>
     </figure>
-
 
-
 
</div>
</div>
-
<div class = "col-sm-6">
 
-
</div>
 
-
<p> The pol genes codes for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. Further the genomes codes for a psi-packaging sequence. This sequence forms a secondary RNA structure and is needed for correct packaging of the viral genome into the virion (3). After proper packaging the virus is leaving the cell and can start the next round of transfection.</p>
 
</div>
</div>
 +
</section>
</section>
-
<h1>Retroviral vectors</h1>
+
<h2 id="Project-TheViralVector-Vectors">Retroviral Vectors</h2>
<section>
<section>
<div class = "row category-row">
<div class = "row category-row">
<div class = "col-sm-6">
<div class = "col-sm-6">
-
<p>Retroviral vectors are used for the delivery of variable gene cargo into cells, a process termed transduction. To ensure safe handling the cis- and trans-acting gene elements are separated from each other. Helper viruses or packaging cell lines, carrying the trans-acting gene-elements are needed for producing the functional viral vector. Besides safety issues this approach also has the advantage that, due to the deletion, a larger gene cargo (~ 8 kB) can be integrated into the vector (4). Our MuLV vector needs the Phoenix eco cell line in order to replicate.</p></div>
+
<p>Retroviral vectors are used to deliver variable gene cargos into cells, a process termed transduction (Fig. 1). The viral vector recognizes a specific receptor on the surface of the cells allowing its internalization and integration into the host genome.
 +
To ensure <a href="https://2014.igem.org/Team:Freiburg/HumanPracticeAndSafety/Safety/viralvector">safe handling</a> in the production of viral particles the cis- and trans-acting gene elements are separated from each other. All the structural proteins (gag, pol and env) needed in order to package a new virus is encoded by a helper plasmid within the packaging cell. The genes are under the control of non-MuLV promoters in order to minimize the probability of a recombination event that would lead to the production of self-replicating viruses.
 +
 +
</p>
 +
</div>
<div class = "col-sm-6">
<div class = "col-sm-6">
</div>
</div>
-
<p> They carry the trans-acting gene elements under non-MLV promoters in order to minimize the probability of a recombination event that would lead to the production of replication efficient viruses. The advantages of retroviral vectors are especially the stable integration of gene cargo and their high specificity but also their flexible genome.</p>
+
<p> The gene of interest is located, together with the psi packaging sequence, on the viral vector plasmid.
 +
  Within the packaging cell line structural genes required for assembly of a new virus are transcribed and translated by the helper plasmid. The viral vector plasmid gets also transcribed but lacks any of the structural genes. The end result is a replicative incompetent recombinant retrovirus that includes the transgene, but lacks any of the structural genes to form a new virus in the host target cell [8].
 +
The advantages of retroviral vectors are especially the stable integration of genes and their high specificity but also their flexible genome.</p>
</div>
</div>
</section>
</section>
-
<p>References</p>
+
<div class="row category-row">
-
<p align="left">1 - The IN Protein of Moloney Murine Leukemia Virus Processes the Viral NA Ends and Accomplishes Their Integration In Vitro</p>
+
<div class="col-sm-6">
-
<p>&nbsp;</p>
+
<div class="container-fluid" style="float: left">
-
<p align="left">2- Integration of murine leukemia virus DNA depends on mitosis</p>
+
<div style="position: relative; float: right; margin-top: 4px;">
-
<p>&nbsp;</p>
+
<a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">Go back to The Receptor</div>
-
<h1>3- NMR Structure of the 101-nucleotide Core Encapsidation Signal of the Moloney Murine Leukemia Virus</h1>
+
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
-
<p>&nbsp;</p>
+
</div>
-
<h1>4 - The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery.</h1>
+
</div>
-
<p>&nbsp;</p>
+
<div class="col-sm-6">
-
<h2>5 - Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics</h2>
+
<div class="container-fluid" style="float: right">
-
<p>&nbsp;</p>
+
<div style="position: relative; float: left; margin-top: 4px;">
-
<p>6 &ndash; Retroviral integration:</p>
+
<a href="https://2014.igem.org/Team:Freiburg/Project/Vision">Read more about Mammalian Systems for iGEM</div>
-
<p align="left">Structure of the initial covalent product andn its</p>
+
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
-
<p align="left">precursor, and a role for the viral IN protein</p>
+
</div>
-
<p>&nbsp;</p>
+
</div>
 +
</div>
 +
 
 +
<section>
 +
<h2 id="Project-TheViralVector-References">References</h2>
 +
<ol class="two-columns small">
 +
<li>Craigie R, Fujiwara T, Bushman F. The IN Protein of Moloney Murine Leukemia Virus Processes the Viral NA Ends and Accomplishes Their Integration In Vitro. Cell. 1990 Aug 24;62(4):829-37.</li>
 +
<li>T Roe, T C Reynolds, G Yu,  P O Brown. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. May 1993; 12(5): 2099–2108.</li>
 +
<li>D'Souza V, Dey A, Habib D, Summers MF. NMR Structure of the 101-nucleotide Core Encapsidation Signal of the Moloney Murine Leukemia Virus. J Mol Biol. 2004 Mar 19;337(2):427-42.</li>
 +
<li>Donald S Anson. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet Vaccines Ther. 2004; 2: 9.</li>
 +
<li>Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med. 2001 Jan;7(1):33-40.</li>
 +
<li>Brown PO, Bowerman B, Varmus HE, Bishop JM. Retroviral integration: Structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525-9.</li>
 +
<li>Balliet JW and Bates P. Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles. J Virol. Jan 1998; 72(1): 671–676.</li>
 +
<li>http://oehs.vcu.edu/chemical/biosafe/lentiviralvectors.pdf</li>
 +
</ol>
 +
</section>

Latest revision as of 03:55, 18 October 2014

The AcCELLerator

The Viral Vector

Retroviral Introduction

Retroviruses are enveloped viruses that are found in fish, avian and mammals. The diameter of a retroviral virion is approximately 80 – 100 nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located (Fig. 2). They are positive-sense and approximately 7 – 11 kb long [5]. The retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Moloney Murine Leukemia Virus (MuLV), which is the origin of the viral vector we are using for our project.

Fig.1: Steps in viral transduction.

Retroviral Tropism And Infection Mechanism

Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect [1]. The Moloney Murine Leukemia Virus (MuLV) is an ecotropic gamma-retrovirus; it only infects rodent cells (mice and rat). This tropism of the virus is granted by its high specificity towards the mCAT-1 receptor [7]. Once attached to the receptor the MuLV enters the target cell and the single stranded viral RNA is transferred into the cytoplasm.

Within the cell a complementary strand of DNA is created by the viral reverse transcriptase, which is then replicated to form a double strand DNA (dsDNA). During mitosis the dsDNA can enter the nucleus due to the breakdown of the nuclear membrane. Once in the nucleus the viral DNA is further processed by the viral Integrase that recesses the the 3’ ends of the DNA by 2 bp. Double stranded viral DNA is then integrated at random into the host genome and, termed as provirus, it starts to express viral enzymes and proteins.

The Retoviral Genome

The MuLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. These so called trans-gene elements are flanked by long terminal repeats (LTR). The 5 ’ LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3’ LTR is needed for polyadenylation of the proviral mRNA. The gag (group specific antigen) genes are precursor polyproteins that form the major components building up the core particle, RNA binding proteins and the nucleoprotein core particle. The pol genes encode for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. The retroviral psi-packaging sequence is a cis-acting RNA element which allows the transcribed viral RNA to be incorporated into the assembly of the new virus [3]. After proper packaging the virus is leaving the cell and can start the next round of infection.

Fig.2: Schematic retrovirus.

Retroviral Vectors

Retroviral vectors are used to deliver variable gene cargos into cells, a process termed transduction (Fig. 1). The viral vector recognizes a specific receptor on the surface of the cells allowing its internalization and integration into the host genome. To ensure safe handling in the production of viral particles the cis- and trans-acting gene elements are separated from each other. All the structural proteins (gag, pol and env) needed in order to package a new virus is encoded by a helper plasmid within the packaging cell. The genes are under the control of non-MuLV promoters in order to minimize the probability of a recombination event that would lead to the production of self-replicating viruses.

The gene of interest is located, together with the psi packaging sequence, on the viral vector plasmid. Within the packaging cell line structural genes required for assembly of a new virus are transcribed and translated by the helper plasmid. The viral vector plasmid gets also transcribed but lacks any of the structural genes. The end result is a replicative incompetent recombinant retrovirus that includes the transgene, but lacks any of the structural genes to form a new virus in the host target cell [8]. The advantages of retroviral vectors are especially the stable integration of genes and their high specificity but also their flexible genome.

References

  1. Craigie R, Fujiwara T, Bushman F. The IN Protein of Moloney Murine Leukemia Virus Processes the Viral NA Ends and Accomplishes Their Integration In Vitro. Cell. 1990 Aug 24;62(4):829-37.
  2. T Roe, T C Reynolds, G Yu, P O Brown. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. May 1993; 12(5): 2099–2108.
  3. D'Souza V, Dey A, Habib D, Summers MF. NMR Structure of the 101-nucleotide Core Encapsidation Signal of the Moloney Murine Leukemia Virus. J Mol Biol. 2004 Mar 19;337(2):427-42.
  4. Donald S Anson. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet Vaccines Ther. 2004; 2: 9.
  5. Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med. 2001 Jan;7(1):33-40.
  6. Brown PO, Bowerman B, Varmus HE, Bishop JM. Retroviral integration: Structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525-9.
  7. Balliet JW and Bates P. Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles. J Virol. Jan 1998; 72(1): 671–676.
  8. http://oehs.vcu.edu/chemical/biosafe/lentiviralvectors.pdf