Retroviruses Flashcards
What are retroviruses and why are they particularly interesting?
Retroviruses are important human and animal pathogens and are particularly interesting because of their unusual replication cycle.
Retrovirus are enveloped viruses with a single stranded RNA genome.
What do retroviruses package and why?
Retroviruses package reverse transcriptase enzyme in the particle to copy their RNA genome into double stranded DNA.
Retroviruses also package an integrase enzyme and integrate their DNA into the host cell’s DNA.
This allows the retroviral genome to be transmitted to both daughter cells when the infected cell divides.
When does the host cell RNA polymerase transcribe the virus’ genome?
During its replication cycle (and also produces the genome for the new virus particles).
Give an example of a retrovirus that belongs tothe genera of Lentiviruses?
Human Immunodeficiency Virus (HIV)
Where did HIV originate?
HIV is very similar to a virus called SIV (simian immunodeficiency virus), which is found in monkeys.
SIV likely jumped species to humans in the late 19th or early 20th century.
HIV-1 evolved from a SIV that infected chimpanzees whereas HIV-2 evolved from a SIV that infected sooty mangabeys.
How many types of HIV are there?
There are two major types of HIV, namely HIV-1 and HIV-2.
HIV-1 and HIV-2 are genetically distinct, sharing only a 55% sequence identity.
What are the subtypes of HIV-1 and HIV-2?
Each type of HIV can be further broken down into groups, which themselves can be subdivided into clades (i.e., subtypes).
HIV-1 comprises groups M (main), O (outlier), and N (non-M or O).
There are two main HIV-2 subtypes, A and B.
What is the most common form of HIV?
HIV-1 is the most common type of HIV; 95% of people living with HIV globally have HIV-1.
Describe the structure of HIV.
HIV is an enveloped virus that has a complex structure.
The genome is in the form of a nucleocapsid, and is packaged within the virus capsid along with reverse transcriptase and integrase.
The matrix protein lies between the capsid and the viral envelope.
Two viral envelope proteins are present; SU (gp120) binds to the CD4 receptor and a chemokine receptor, and TM (gp41) is the fusion peptide that results in the viral envelope fusing with the cell’s plasma membrane, thus releasing the capsid into the cell’s cytoplasm.
What does the HIV virus envelope contain?
The virus envelope contains a trans-membrane glycoprotein (TM, gp41) that is joined to a second virus glycoprotein (SU, gp120) by a di-sulphide bond.
What part of HIV binds to the host cell protein CD4?
The gp120 binds to the host cell protein CD4 and the gp41 is the fusion protein.
What lines the inner surface of the envelope in HIV?
The matrix (MA, p17) protein
What is inside the HIV envelope?
A conical capsid made from the virus protein p24 (CA).
What does the HIV capsid contain?
Two (+) sense RNA molecules that are covered with nucleocapsid (NC, p7) proteins, and the enzymes involved in the early stages of genome replication, reverse transcriptase (RT) and integrase (IN).
Protease proteins can be found inside and outside of the capsid.
Vpr is a regulatory protein.
The genome of HIV has several features that are common to retroviruses and distinct from other RNA viruses.
What are they?
The genome is diploid: it consists of two, usually identical molecules of single-stranded RNA.
Both RNA strands are (+) sense with a 5’ cap and a 3’ polyA tail, but it does not serve as a mRNA immediately upon entry into the host cell cytoplasm.
The genome is associated with a specific tRNA (derived from the host cell) that is used to prime the synthesis of DNA.
Finally, the genome is synthesized and processed by the host cell mRNA “handling machinery” after the virus has integrated its DNA into the host cell chromosome in the previous round of infection
What are the three major genes that HIV has that are common to all retroviruses?
HIV has three major genes (gag, pol and env) that are common to all retroviruses.
What are the genes that HIV has that code for regulatory and accessory proteins?
HIV has six genes that code for regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) proteins.
What is the gene order in all Retroviruses?
The gene order in all Retroviruses is invariant:
5’ – gag– pol– env– 3’.
Describe the HIV genome.
The genome of a HIV consists of two identical (+) RNA molecules.
Although these resemble mRNA molecules, they are not translated by the ribosomes when the virus infects the cell.
The gene order for all retroviruses is 5’ – gag – pol – env – 3’.
The gag gene encodes structural proteins, the pol gene encodes the enzymes needed in genome replication, and the env gene encodes the envelope proteins needed to bind to host cells.
Note: This diagram is showing the DNA structure after the process of reverse transcription
What does the gag gene encode?
The gag gene encodes the Gag polyprotein, that when cleaved yields the CA, MA and NC proteins.
What does the pol gene encode?
The pol gene encodes the Pol portion of the Gag/Pol polyprotein, that when cleaved yields the enzymes PR, RT and IN.
The PR becomes activated after the assembly of the virus particle and cleaves the Gag and Gag/Pol polyproteins at specific sites.
What does the env gene encode?
The env gene encodes the Env polyprotein, that when cleaved yields the gp120 and gp41 proteins.
Reverse transcriptase is an enzyme unique to Retroviruses and has at least three different enzymatic activities.
What are they?
- an RNA dependent DNA polymerase,
- RNase (an enzyme to degrade RNA), and
- a DNA dependent DNA polymerase.
What are the key points in the process of reverse transcription of the RNA genome in HIV?
- RT uses a tRNA as a primer to synthesize a DNA copy of the RNA genome. This creates a DNA:RNA hybrid molecule,
- RNase H removes most of the RNA strand (this particular form of RNase can digest the RNA in hybrid molecules),
- the remaining RNA is then used as a primer for synthesis of the complementary strand of DNA,
- the RNA has been copied to yield a double stranded DNA molecule.
When does the process of reverse transcription occur?
In the capsid after the capsid has been released into the cytoplasm.
What is the primer binding site?
Part of the tRNA molecule hybridizes to a complementary site on the RNA genome called the primer binding site (PBS).
What direction does reverse transcriptase read the sequence of genomic RNA?
What direction does it add the complementary DNA nucleotides?
The RT reads the sequence of the genomic RNA in a 3’ to 5’ direction, adding the complementary DNA nucleotides in a 5’ to 3’ direction.
What happens when the RT reaches the end of the RNA strand?
RNase H activity digests this portion of the RNA template.
The DNA product is transferred to a site near the 3’ end of the RNA template.
The newly made minus-strand of DNA binds to the repeat (R) sequence on the RNA template.
RT reads the sequence of the genomic RNA in a 3’ to 5’ direction, adding the complementary DNA nucleotides in a 5’ to 3’ direction to complete the synthesis of the minus-strand of DNA.
What is the poly-purine tract?
What is it used for?
Most of the RNA is digested, except for a small region called the poly-purine tract (PPT).
Using the PPT on the RNA strand as a primer, the RT reads the minus-strand DNA as a template in a 3’ to 5’ direction, adding the complementary DNA nucleotides in a 5’ to 3’ direction.
RNase H activity digests this PPR region of the RNA template.
The newly made fragment of plus-strand DNA is transferred to the 3’ end of the minus-strand DNA, and the RT then completes the synthesis of the double stranded DNA
How are the long terminal repeats generated?
The long terminal repeats (LTRs) are generated as a consequence of the reverse transcription process.
What is the end result of reverse transcription of the HIV genome?
The R-U5 (repeat sequence unique sequence 5’ end) and U3-R (unique sequence 3’ end repeat sequence) from the genomic RNA have now both become U3-R-U5 in the viral DNA, giving it the required LTR at each end.
The R site in the 3′ LTR contains a cleavage site and a polyadenylation site used for the transcription of viral mRNA.
Describe the process of reverse transcription.
- The tRNA binds to a complementary sequence in the viral genome and is used as a primer by the RNA dependent DNA polymerase (RDDP) activity of reverse transcriptase (RT) to synthesize complementary DNA (the (–) strand).
- The RNase H activity of RT removes the short stretch of the 5’ end of the viral RNA.
- The short DNA sequence can bind to the 3’ end of the other strand of the RNA.
- The RDDP of RT uses this RNA strand as a template to continue DNA synthesis.
- The RNase H continues to remove RNA leaving short segments to be used as primers for DNA synthesis.
- The DNA dependent DNA polymerase (DDDP) of RT then uses the RNA primers to synthesize the (+) sense DNA strand.
- The key point in this process is that the single stranded RNA molecule is used as a template to synthesize double stranded DNA molecules.
Where does the HIV DNA synthesis take place?
In a protein-coated core inserted into the cytoplasm of the host cell.
Where does HIV get the primer needed to synthesize the first strand of DNA?
How about the primer for the second strand?
The primer for the first strand of DNA is a packed tRNA molecule, and the primer for the second strand of DNA is a small piece of RNA left behind after the RNase action of the RT degrades the RNA part of the RNA:DNA hybrid.
The genome of the HIV particle is composed of a double stranded RNA – one strand is positive sense and one strand is negative sense.
True or false?
False.
HIV is an enveloped virus.
True or false?
True.
Human immunodeficiency viruses have a dsRNA genome.
True or false?
False.
HIV requires a co-receptor to infect cells.
True or false?
True
The genome of the HIV is reverse transcribed in the particle before it enters into the host cell.
True or false?
False.
In reverse transcription the first strand of DNA synthesized is a plus strand.
True or false?
False.
RNAse H is used to degrade host cell RNA such that translation of viral mRNA transcripts is priority for the cytoplasmic ribosomes.
True or false?
False.
Reverse transcriptase is an RNA dependent DNA polymerase.
True or false?
True.
Retroviruses package a positive ssRNA genome, which resembles an mRNA and is immediately translated upon entry into the host cell.
True or false?
False.
Unlike most other viruses, retroviruses package two identical copies of their genome in the virion.
True or false?
True.
HIV has 3 main genes; gag, pol and env.
True or false?
True.
Retroviruses package reverse transcriptase enzyme and integrase in the virus particle.
True or false?
True
Reverse transcription results in a single strand of (+) RNA being copied into a double stranded DNA structure.
True or false?
True.
Reverse transcriptase has two distinct polymerase activities: RNA dependent DNA polymerase and DNA dependent DNA polymerase.
True or false?
True.
RNase H is a nuclease that degrades all of the RNA in the cell.
True or false?
False.
RNase H is one of the functional active sites in reverse transcriptase.
True or false?
True.
The reverse transcriptase uses a cellular mRNA as a primer to make the first strand of DNA.
True or false?
False.
The reverse transcriptase does not require a primer to make a DNA strand.
True or false?
False.
What are lentiviruses?
Slow virus
Infections with lentiviruses are characterized by a long interval between the initial infection and the onset of serious clinical symptoms.
eg - HIV
What is AIDS?
Acquired Immune Deficiency Syndrome
AIDS is the final stage of the HIV infection
Acquired – means that the disease is not hereditary but develops after birth from contact with a disease-causing agent (in this case, HIV).
Immunodeficiency – means that the disease is characterized by a weakening of the immune system.
Syndrome – refers to a group of symptoms that indicate or characterize a disease.
How does HIV damage a person’s immune system?
Both types of HIV damage a person’s immune system by destroying the CD4 T cells.
Recall how crucial T helper cells were to the adaptive immune responses.
When will a person that has HIV+ status be diagnosed with AIDS?
When they have one or more opportunistic infections (or certain cancers) and a CD4 T cell count of less than 200 cells per µl of blood.
Where does HIV get the tRNA that associates with the two strands of positive sense RNA?
Acquired from the host cell of the previous infection
After infecting the cell, the first step in the HIV replication cycle involves translation of the RNA on a ribosome.
True or false?
False
When HIV first enters into the cytoplasm, it is not completely uncoated.
The reverse transcription occurs in the core of the virus, and the core delivers the dsDNA to the nucleus.
What is the group specific antigen gene?
gag
Encodes capsid, nucleocapsid, matrix, protease
What is the polymerase gene?
pol
Encodes reverse transcriptase, integrase
What does the env gene encode?
encodes the viral envelope glycoproteins - gp120 and gp41
What mediates the conversion of the RNA genome into a ds DNA genome?
The reverse transcriptase (RT) enzyme.
The resulting dsDNA product from reverse transcription is slightly longer in length than the original RNA due to the RT process.
This produces a promoter and transcription terminator in the DNA sequence.
True or false?
True
What are the main steps of reverse transcriptase that Dr. Kion wants you to know?
- RNA dependent DNA polymerase activity of reverse transcriptase (Reads the RNA template and synthesizes a complementary DNA strand)
- RNase activity of reverse transcriptase (degrades the RNA strand)
- DNA dependent DNA polymerase activity of reverse transcriptase (Reads the DNA template and synthesizes a complementary DNA strand to make dsDNA)
Reverse Transcriptase has 3 distinct enzymatic activities, each catalyzed at a different site in the enzyme.
Which of the three activities in the Reverse Transcriptase enzyme is the most suitable for targeting for an anti-viral therapy and why?
RNA dependent DNA polymerase
RNA-dependent DNA polymerase is a unique virus activity that makes it ideal for anti-viral therapy.
Something that targets the other enzymatic functions of RT would be terrible – it would impact non-infected cells too (and interfere with DNA synthesis).
How do retroviruses leave the host cell permanently infected?
Retroviruses copy their RNA genome into double stranded DNA and then integrate the DNA into the host cell’s DNA, leaving the host cell permanently infected.
The virus uses the host cell’s RNA polymerase II and splicing enzymes to produce its mRNAs and its genome.
How does the replication cycle begin for HIV?
The viral glycoprotein gp120 binds to the CD4 cell surface marker of susceptible cells (usually T helper cells, but macrophages and dendritic cells are usually the first cells infected).
The binding to CD4 alone is not sufficient for the virus to infect the cell.
Describe HIV attachment and entry of the virus capsid.
HIV requires a receptor and a co-receptor.
The binding of gp120 to the CD4 protein induces a conformation change in both gp120 and gp41 that ultimately results in the fusion of the virus envelope with the host cell plasma membrane and result of the capsid into the cytoplasm.
Describe the delivery of the HIV genome to the cell nucleus.
The virus reverse transcribes its RNA genome into the double stranded DNA genome.
Following arrival at the nucleus, the pre-integration complex (PIC, which contains the provirus and integrase) traffics through the nuclear pore complex (NPC).
Describe the integration of proviral DNA into the host cell genome.
The integrases recognizes specific sequences and uses its endonuclease activity to make a staggered cut into the host cell DNA.
The integrase transfers the proviral DNA so that it is joined to the host cell DNA.
Host cell DNA polymerases repair the single stranded DNA of the target site.
The process of integration results in the duplication of the target site.
Describe the mRNA of HIV.
The integrated provirus DNA is transcribed and processed by host cell enzymes to at least seven different mRNA.
The thin lines in the diagram represent the RNA that was spliced out of the primary RNA.
The thicker purple lines represent the remaining RNA.
The blue dot represents the 5’ cap.
Describe the HIV-1 frameshift site.
Two cis-acting RNA elements (i.e., they are on the same strand) are separated by a single-stranded spacer.
These act with trans-acting factors to regulate post-transcriptional gene expression.
Describe the mechanism of translation to merge proteins encoded by two overlapping open reading frames.
Ribosome frameshifting
For HIV, the frameshift occurs at low frequency and consists of ribosomes slipping by one base in either the 5’(-1) direction during translation.
Other virus species may have the ribosome slips in the 3’(+1) direction, and some virus species contains both a +1 and a -1 ribosomal frameshifts.
Describe HIV assembly and egress.
HIV Gag protein drives virus assembly and recruits the ESCRT proteins for facilitate virus egress.
The ESCRT pathway is used in cellular processes such as endocytosis where the membrane constricts away from the cytoplasm and membrane fission is catalyzed by cytoplasmic dynamin, which acts from the outside of the bud neck.
When the viruses bud, the membrane must be constricted toward the cytoplasm, and cytoplasmic host factors that catalyze membrane fission must work from within the bud neck.
The ESCRT pathway can perform such “reverse topology” membrane fission events.
Describe the maturation of HIV.
The maturation of HIV to form an infectious particle involves cleavage of the polyprotein and several conformational changes.
Note the morphologically distinct capsid.
The cleavage of the Gag-Pol protein results in the production of a functional reverse transcriptase and integrase.
The enzymes, however, remain inactive until the virus enters the cytoplasm of a host cell and gains access to nucleotides to allow for the process of reverse transcription followed by integration.
How is HIV-1 transmitted?
Through the direct contact of a mucus membrane or the bloodstream with a biological fluid containing HIV-1.
These biological fluids include blood, semen, vaginal fluid, pre-ejaculate fluid and breast milk.
What is HIV not transmitted by?
air, water, sweat, tears, insects, pets, contact with toilet seats.
Describe the acute stage of HIV infection.
This stage lasts on average about four weeks.
The symptoms of infection are non-specific and therefore are often not recognized as signs of HIV-1 infection.
Patients are frequently misdiagnosed with a more common illness such as Influenza.
During this time, there is an abundance of virus in the blood and its dissemination to lymphoid organs.
The acute infection stage is also marked by a drop in the number of circulating CD4 T lymphocytes.
The body’s immune system activates CTLs that kill some virally infected cells (resulting in the decline in CD4 T cells), as well as activates some B cells resulting in antibody production.
Antibodies against several HIV protein can be detected (i.e., anti-gp120 antibodies).
Describe the AIDS stage of HIV infection.
The definition of AIDS has changed over time as the understanding of the virus and its relationship with the immune system increases.
When CD4 T cell numbers decline below a critical level (~200/mm3), HIV-1-positive individuals become highly susceptible to infections with a variety of opportunistic bacteria, viruses, fungi and parasites that are normally controlled by the elements of the immune system that HIV-1 has damaged.
In this stage, the amount of virus circulating in the blood is usually very high
HIV infection is the same as AIDS.
True or false?
False
How do reverse transcriptase inhibitors work?
RT inhibitors work early in the HIV cycle to prevent the virus from copying its RNA genome into a double stranded DNA copy.
As a result, the cell does not become permanently infected with HIV.
How do protease inhibitors work?
The protease enzyme is required for HIV maturity, allowing the virion to become infective.
These drugs competitively inhibit the HIV-1 protease, preventing maturation of the viruses by blocking the cleavage of the Gag-Pol polyprotein to release RT and integrase.
This results in a non-infectious virus particle.
The virus may be able to bind to another host cell, but the process of reverse transcription will not occur when the capsid enters into the cytoplasm.
These drugs are transition state analogues and can be considered as “plug” drugs
Protease inhibitors work in infected cells and result in the production of defective virus particles.
How do integrase inhibitors work?
Integration of the virus DNA into the host cell genome is a crucial step in the HIV life cycle.
The part of the replication cycle can be blocked by integrase inhibitors.
Integrase inhibitors work early in the HIV cycle to prevent the DNA copy of the genome from inserting itself into the host cell genome.
As a result, the cell does not become permanently infected with HIV.
Compare pre and post exposure prophylaxis.
Individuals that are not currently infected with HIV may take antiretroviral medications to prevent infection if exposed to the virus.
