Viruses and Biotechnology

What is a virus?

• Viruses are intracellular obligate parasites that MUST infect a host cell in order to reproduce. 

• They are not considered to be alive by most scientists because:

  • They don’t grow or develop

  • They don’t metabolize energy

  • They cannot reproduce without the aid of a host cell.

Viral structure

Genetic Material (DNA/RNA) surrounded by a protein shell called a capsid

Envelope

• Cell-membrane like structure that is made of lipids. 

• Surrounds the capsid for extra protection. 

Spike Protein

• Spike shaped projections made of protein 

• Help viruses attach to and infect their host cells 

Capsid

• Protein coat that surrounds  and protects the virus and genetic material.

Genetic Material

• DNA or RNA that carries the instructions for making more copies of the virus. 

Viral Specificity

• The shape of the virus and its spike proteins are specific to the type of host cell they infect to reproduce.

• Viruses are highly specific to their host cell species.

  • Plant viruses only infect plants

  • Animal viruses only infect their species of animal.

    • Some viruses will infect related species

  • Bacteriophages only infect bacteria.

Types of Viruses

Eukaryotic Viruses

-Much more diverse than prokaryotic viruses, with a greater variety in genome type. 

-Genome can be dsDNA, ssDNA, dsRNA, ssRNA, etc. 

• Good examples: Retroviruses (like HIV) and Influenza Viruses 

Prokaryotic Viruses

-Also called bacteriophages or just phages. 

-Much less diverse than eukaryotic viruses with less genome types. 

Retroviruses

• Retroviruses store their genetic material as 3’ to 5’ single stranded RNA (ssRNA)

  • Basically backwards mRNA 

• 3’ to 5’ orientation prevents the RNA from being translated by host cell ribosomes

  • Ribosomes can only translate from 5’ to 3’

• Before retroviruses can reproduce, their ssRNA genome has to be converted into DNA so that 5’🡪3’ mRNA can be made

• Retroviral genomes code for the enzyme reverse transcriptase (RT) to accomplish this task. 

  • Retroviruses are the only organism on earth that codes for this protein. 

• RT is unique because it “violates” the central dogma of biology by copying RNA into DNA. 

Influenza Virus

-RNA virus that is prone to mutations in spike antigen proteins(SAP). Mutations change the amino acid sequence of the SAP leading to new strains of the flu through antigenic drift. 

• Co-infection by two flu strains can cause the two virus strains to combine

  • This is called Antigenic Shift OR

  • Viral Recombination 

• This is what allows the flu virus to cross the species barrier

  • Like with Swine Flu or Avian Flu 

Because of Antigenic shift and drift, it is impossible to have permanent immunity to the flu. 

Bacteriophages

• Bacteriophages only infect prokaryotic organisms. 

  • I.e. bacteria 

• They are typically more simple than eukaryotic viruses

• Phages are an excellent and simple example of the general method viruses use to reproduce. 

Viral Reproduction

• Viruses attach to the host cell and inject their genetic material. 

• Once their genetic material is inside, the conditions inside the cell will tell the virus what type of replication it should complete

• Two Replication Options: 

  • Lytic Cycle

    • Virus infects the host cell and uses host organelles to make more copies of itself. This is very fast! 

    • Infected cells explode and die during the lytic cycle to release the virus. This causes sores to appear where the cells died.

  • Lysogenic Cycle 

    • No new viruses are made during the lysogenic cycle. The viral DNA is dormant and is copied every time the host cell replicates. 

    • Virus inserts its DNA into the host cell, and the DNA adds itself into the host cell DNA. 

Genetic Variation in Viruses
Viruses evolve quickly, and often faster than their hosts. How?

1. Viruses, just like cells, can acquire mutations. 

• Just like other cells, a large majority of these mutations occur from mistakes that are made when their genomes are replicated inside host cells. 

• DNA viruses are replicated using the cell’s DNA polymerase enzymes, which have proofreading functions. 

• RNA viruses must use their own special enzymes called RNA-dependent RNA polymerases to make copies of their genetic material. These polymerases generally do not have a proofreading function and end up making many more mistakes as a result. Thus, RNA viruses generally mutate much faster than DNA viruses. 

2. Viruses reproduce very quickly, and with high mutation rates, many new genetic variants can arise just from mutations alone. 

3. Viruses can also undergo recombination, a process that happens when similar viruses infect the same cell and their genetic material ends up packaged together in new viruses being made. 

   • Ex: influenza viruses. 

Biotechnology

• Genetic engineering techniques can be used to analyze and manipulate DNA and RNA. These include:

  • Polymerase Chain Reaction (PCR)

  • Electrophoresis

  • Bacterial Transformation

Polymerase Chain Reaction(PCR)

• Polymerase chain reaction (PCR) is a widely used method that rapidly makes millions to billions of copies of a specific DNA sample in a test tube. 

  • This allows a very small sample of DNA to be amplified to a large enough amount to study in detail.

• PCR requires the following components: the DNA sample to be amplified; DNA primers, DNA nucleotides, and Taq polymerase.

  • Taq polymerase is a thermostable polymerase isolated from a heat-tolerant bacteria (Thermus aquaticus). It is able to function without denaturing at high temperatures.

• PCR involves repeated cycles of heating and cooling that allow DNA to be synthesized.

• The basic steps of PCR are:

1. Denaturation (96°C): Heat the reaction to separate, or denature, the DNA strands. This creates single-stranded template DNA.

2. Annealing (55°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.

3. Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new DNA strands.

4. Repeat!

Gel Electrophoresis

• Review the Restriction Enzyme Lab we did in class.

• Gel electrophoresis separates molecules by their size and charge.

  • Type of molecules that can be used in electrophoresis: DNA, RNA, and proteins.

• Molecules are “loaded” into wells in the gel. Then an electrical current pulls the molecules through the gel.

• Molecules will travel in the gel at different speeds (thus taking them different distances) based on their:

  • Size: smaller molecules travel faster than larger ones

  • Charge: stronger charged molecules travel faster than weaker charged molecules

• In DNA gel electrophoresis, DNA samples are cut using restriction enzymes before being ran through a gel, producing DNA fragments of different sizes.

• Restriction enzymes are proteins that cut DNA at specific sequences, called restriction sites.

  • Ex: The restriction enzyme EcoRI cuts at the restriction site: GAATTC

• DNA is pulled towards the positive cathode, because DNA is negatively charged. Smaller DNA fragments travel further than larger fragments. 

• One application of DNA gel electrophoresis is to determine what alleles are carried by an individual (their genotype). 

• Different alleles for a gene can have different restriction sites that produce different sets of fragments when cut by restriction enzymes. 

• Another application of DNA gel electrophoresis is to determine a child’s mother/father.

• A child has DNA from both parents. The fragments produced when a child’s DNA is digested (cut) by restriction enzymes will be a combination of fragments found in the mother and father. 

• DAD 3 IS THE FATHER!

Biotechnological Applications of Restriction Enzymes

• Scientists have isolated RE’s from many different species of bacteria and identified the specific sequences that they cut. 

• From there, they use RE’s like molecular scissors to “cut” DNA that have those sequences. 

• This is incredibly useful for gene cloning,  research and DNA testing purposes. 

How Restriction Enzymes Work

• Restriction enzymes recognize specific DNA sequences.

• When they encounter these sequences, they “cut” both strands of the DNA by breaking the covalent bonds between the nucleotides within a strand. 

  • This is called a double stranded cut.

Biotechnology Technique: Restriction Enzyme Digest

-Lab protocol used to cut DNA into fragments using RE’s

• Incubate DNA sample with pre-determined RE’s (usually overnight)

• RE’s cut the DNA, creating fragments.

• DNA fragments can be put into vectors/plasmids and used to make bacteria produce the gene product of that DNA sequence through bacterial transformation  

• OR You can run the  products RE Digest on a agarose gel and compare it to a ladder to see the banding pattern. 

Biotechnology Technique: Bacterial Transformation

Lab protocol that causes bacteria to take in and produce proteins from non-bacterial DNA found in vectors/plasmids. 

• Incubate vector/plasmid with bacteria that have been heat shocked to open up pores in their membrane. 

  • Vector/plasmid will have an antibiotic resistance gene in it. 

• Bacteria take up the plasmids with your gene of interest in it. 

• Grow colonies of these bacteria on nutrient media that contains antibiotics

  • Only bacteria with the plasmid will survive. 

• Bacteria use their own protein synthesis machinery to make the gene product of your gene. 

• This is how they make insulin for diabetes patients. 
  
  

DNA Sequencing

• DNA sequencing determines the order of nucleotides in a DNA molecule. There are several techniques, but the Sanger sequencing technique is the simplest. 

• This technique requires special “chain-terminating” nucleotides called dideoxynucleotide triphosphates (ddNTPs), which are missing the important 3’ hydroxyl group needed to make a phosphodiester bond with other nucleotides.

  • Normal DNA nucleotides are called deoxyribonucleotides triphosphates (dNTPs), which are assembled together by DNA polymerase to synthesize a DNA strand. 

• Copies of the DNA molecule to be sequenced, along with primers, DNA polymerases, normal nucleotides (dNTPs), and fluorescently-labeled chain-terminating nucleotides (ddNTPs) are used together to replicate the DNA strand.

  • The different types of ddNTPs (adenine, guanine, cytosine, and thymine) are fluorescently labeled different colors to distinguish them.

• If DNA polymerase uses one of the ddNTPs while replicating a DNA strand, the elongation of that strand will stop. Thus, strands will be copied at different lengths, depending on how far DNA polymerase copies the strand without using a ddNTP.

• All of these varied-lengthed copies can be ran through gel electrophoresis to sort them by size.

• Their fluorescent color will indicate what nucleotide was at the last position on each strand. 

• The sequence of the DNA molecule can then be determined using the data.