Retroviruses Flashcards
What is the structure of the virus particle?
These viruses have a + sense RNA genome and include HIV as well as HTLV-1. They have an icosahedral structure and are enveloped. They have untranslated regions at the 3’ and 5’ ends.
What are the gag, env and pol gene products?
- The env gene products include a transmembrane region (TM) as well as surface glycoproteins (SU) that extend out of the side of the surface of the viral particle. These arise from spliced mRNA.
- Gag encodes for the matrix (MA), capsid (C) and nucleocapsid (NC) products. The icosahedral capsid contains two copies of the RNA and either one can serve as the template to produce new copies of the genome. These are the structural genes.
- The pol gene encodes for reverse transcriptase which is used to convert the RNA genome into a DNA copy and this is called the pro-viral DNA. We also have integrase which is an enzyme that is used for integration of the DNA copy into the host genome. These are the viral enzymes.
What is the structure of the genome of the virus?
- There are two copies of the (+)ssRNA genome and so it is a diploid virus that can undergo recombination between the two strands to diversify its genetic material. There are also two copies of annealed transfer RNAs. These RNAs are capped at the 5’ end and polyadenylated at the 3’ end so that they can serve as mRNAs to synthesise proteins.
How are pol gene products produced?
This is through a frameshift mutation. It is the full-length genome mRNA that makes both gag proteins and pol proteins and it does so by a frameshift mutation that occurs 1 in every 20 translations to 1 in every 5 translations. The reading frame of Pol is shifted back -1 of the preceding Gag frame. There is a polyU sequence and a structured RNA that causes the ribosomes to stutter and stall - and shift reading frames.
- You have a polyprotein that joins gag to the polymerase polyprotein -> produces a gag-pol polyprotein.
How do retroviruses enter cells?
A site on the envelope SU protein binds to a cell receptor and this interaction causes a conformational change in the TM protein that allows a hydrophobic fusion sequence to fuse the virion membrane and a cell membrane. Most retroviruses fuse their membrane with the plasma membrane of the cell and release the capsid.
What is the process of reverse transcription?
In the cytoplasm, dNTP’s enter the core and the reverse transcriptase enzyme converts the (+)ssRNA genome into a (-) strand of DNA. The primer for synthesis of the (-) DNA is the tRNA bound to the genome. The (+) DNA strand is then made from this and this is a copy (cDNA).
What is the process of integration.
The dsDNA is then delivered to the nucleus where the integrase enzyme catalyses its random integration into the host DNA. Within this location, it is called the provirus and this is the template to produce the viral RNA’s.
What do the viral RNA’s code for?
These viral RNAs are initially full length genome RNAs that encode for the gag polyproteins or the gag-pol polyprotein. These proteins move and assemble onto the plasma membrane, coordinating with the translation products from spliced RNA to produce the envelope proteins that translate in the ER and golgi network -> they are heavily modified by glycosylation and protein folding to make these mature fibres that then traffick to the plasma membrane and assemble together with the gag, gag-pol polyproteins forming new virus particles. They bud from the infected cell without killing the cell and then undergo a maturation step where the protease enzyme cleaves the polyproteins into their individual components making fully infectious viral particles.
What are the steps of reverse transcription?
The transfer RNA is able to bind to the primer binding sequence (PBS). The tRNA from the cell provides the primer against the template here at the end of the RNA. The RNA has a repeated sequence at either end as well as a U3 and U5 region.
The U5R is transcribed and then once the RNA is cleaved for this section by RNase H activity, it is able to jump and relocate against the repeated RNA sequence at the 3’ end. It commences an elongation of the negative sense strand of DNA. With further cleavage of the RNA template, we end up with a small segment of RNA remaining called the polypyrimidine track sequence that can serve as primer for the reverse transcriptase enzyme to elongate against the negative sense DNA to produce the + sense DNA. In order to elongate the + sense DNA strand complementary against the primer of tRNA. This can once again reposition down to the copied negative sense PBS of the DNA to form a new primer template combination to elongate and complete the provirus. This provirus now has duplicated sequences at the 5 and 3’ ends to make a LTR structure.
What does the proviral DNA look like?
There is a duplication of the untranslated sequences (R, U5 and U3) at the ends of the genomic (+)ssRNA and it generates a dsDNA structure called the long terminal repeat or LTR that is common in mammalian DNA. This is a highly error prone process generating mutations (1 error in every 10,000 nucleotides) and the RT enzyme can switch between the two different genomes to generate a recombinant retrovirus genome.
Is provirus integration site specific?
There is no site specific integration and most integration occurs in transcriptionally active DNA. Integration is required for production of the new virus and simple retroviruses (viruses other than HIV) cannot integrate their cDNA into terminally differentiated cells as they require cell division. There is some protein production from circular and linear proviral cDNA.
What is the process of proviral genome integration?
This occurs randomly and retroviral genomic RNA is expressed from the integrated proviral DNA genome. The integrase enzyme binds to specific sequences within the LTR and then the cleavage event leads to replication and duplication of a small number of sequences, usually 2-4 nucleotides but can become a marker for these integration events when you’re sequencing the genome of the infected cell.
The LTR that forms is the important transcription unit of the virus. The U3 contains the enhancer sequences and the transcription start signals (TAATA), the capping site (the commencement of RNA synthesis) that defines the beginning of the R structure and then with the R at the other end there is a polyadenylation signal that defines the end position of the RNA synthesis and the addition point of polyA tail.
Does our genome contain retroviruses already?
Yes. There are integrated retroviruses known as endogenous retroviruses and they exist as integrated genomes that are transmitted in the germline. Around 8-10% of all of our DNA is made up of retroviruses and they can be full length retroviruses. Endogenous retrovirus elements are known as retroelements and are recognised by flanking repeat sequences that occur during integration.
We have endogenous retroviruses, retrotransposons, retroposons (LINEs), retrosequences (SINEs) and processed pseudogenes.
What is the Koala retrovirus?
This is causing major decline in populations as it is a replication-competent genome and it has been associated with leukemia, neoplasia and chlamydiosis.
Human retroviruses like HTLV-1 infect human cells as well.
How does HIV enter cells?
This enters cells through penetration and this is fusion at the plasma membrane. Attachment is mediated by non-specific engagement with cell surface lectins (DC-SIGN). The HIV-1 envelope glycoproteins gp120/41 bind to the CD4 cell receptor and this induces a conformational change in gp120 that exposes a binding site for the chemokine coreceptor (CCR). Binding of gp120 SU to CCR induces a conformational change in gp41 TM that leads to insertion of the fusion peptide into the membrane.
