8 - Retroviruses

Question 1

What do retroviruses infect? What are the consequences to infection by retrovirus?

Answer 1

Infect insects, fish, humans.

Consequences:

• no ill effects
• tumors: rapid onset or long latency
• wasting diseases, neuro disorders
• immune deficiencies (HIV)

Question 2

What are properties of retroviruses?

Answer 2

Acquire host cells chromosomes - oncogenes

Insert into host cell chromosome - can activate or inactivate genes

Rapid genome evolution via mutations through replication and recombination

Question 3

What are the two major types of retroviruses?

Answer 3

Simple and complex

Question 4

What is the structure of HIV?

Answer 4

Enveloped viruses with envelope protein in membrane and matrix protein under lipid bilayer.

Capsid made of group specific antigens which are products of the gag gene: Matrix, CApside, NucleoCapsid, PRotease.

2 copies of + ssRNA genome, only virus that is diploid and accounts for recombination potential.

Question 5

What are the basic components of a simple retrovirus genome from 5’ (left) to 3’ (right)?

Answer 5

R: repeat (located on each end)
U5: unique to 5’ end
Gag gene: encodes gag proteins which are made as a polyprotein that gets clipped
Polymerase gene: encodes reverse transcriptase and integrase (IN)
Envelope (Env) gene: encodes env protein made as a precursor and gets cleaved into the surface (SU) and transmembrane (TM) domains.
U3: unique to 3’ end
R: on each end

Question 6

The retroviral genomic RNA is made by the hosts RNA Pol II, so they are _______ and ______ like other host pol II mRNAs.

Answer 6

Capped and adenylated

Question 7

What are the components of a complex retroviruses genome?

Answer 7

Similar organization to simple retroviruses with gag/pol/env except numerous additional genes/proteins are present.

These accessory proteins are generated by complex alternative splicing (simple retroviruses do a single splice to make env).

Question 8

What are steps in the first phase of the HIV replication cycle?

Answer 8

1. Adsorption
2. Penetration and uncoating
3. Reverse transcription
4. Transit to the nucleus
5. Integration into host DNA

Question 9

What are the steps in the second phase of HIV replication?

Answer 9

6. Viral RNA synthesis, host pol II
7. RNA processing
8. Virion protein synthesis
9. Assembly and budding
10. capsid maturation (proteolysis)

Question 10

What occurs during the adsorption step of HIV replication? What occurs after this?

Answer 10

Virus binds cell via env protein and host cell receptors.HIV receptor is CD4/CCR5.

Next penetration occurs when viral envelope fuses with the cell membrane either at the cell surface or in an endosome after endocytosis.

Question 11

What occurs after the virus penetrates the host cell?

Answer 11

Uncoating: genomic RNA is only partially uncoated and remains in a protein particle in the cytoplasm (this is to prevent ribosome binding because we want to make dsDNA not translate the RNA right now).

RT, IN, and gag proteins remain associated with incoming genomic RNA. The proteins are needed to convert the ssRNA genome to dsDNA, for nuclear import, and integration.

Question 12

What is the function of reverse transcriptase?

Answer 12

Converts ssRNA to dsDNA in the cytoplasm. This enzyme is carried in with the infecting virion.

Question 13

What are the two polymerase functions of reverse transcriptase? What are characteristics of this enzyme?

Answer 13

RdRp: copies RNA to DNA

DNA-dependent DNA polymerase: copies a second strand of DNA from the first strand

Error prone and accounts for rapid evolution and drug resistance (important target for drug therapy).

Question 14

What carries out integration after the ssRNA is converted into dsDNA? What does it require?

Answer 14

Integrase (IN) protein which enters with the virus and remains associated with the dsDNA.

It requires that the dsDNA has access to the host DNA.

Question 15

How does HIV differ from other viruses in terms of the integration step of replication?

Answer 15

Many retroviruses cannot cross the nuclear membrane and need cell division to integrate.

HIV CAN cross the nuclear envelope.

Question 16

Describe how integrase (IN) works? What happens once the integration has occurred?

Answer 16

Recognizes and is specific for sequence s at the ends of dsDNA (ie U3 and U5) but is NOT specific for host sequences.

IN protein alone performs the insertion (makes it a good target).

Ones integration occurs, the virus is a PERMANENT resident of the host cells DNA.

Question 17

What is the major role of the LTR? What is the orgnaization of LTR?

Answer 17

To direct synthesis of viral RNA.

In the LTR: U3 contains binding sites for cellular transcription factors required for high level RNA synthesis

Question 18

What is the function of U3?

Answer 18

Has signals recognized by the cell’s transcription machinery which directs transcription at the beginning of the “R” region (ie it acts like a promoter).

Despite it being at both ends, only the 5’ (left end) LTR is transcriptionally active (except in special circumstances).

Question 19

What can the spectrum proteins that bind U3 influence? What is an example?

Answer 19

Which tissue/cells a retrovirus is active in (tropism).

HIV LTR requires TF NFkB, which is only expressed in T cells. HIV is not transcribed in infected memory T cells because memory T cells don’t express NFkB.

Question 20

What occurs after pol II transcribes?

Answer 20

The viral RNA is polyadenylated and some is spliced to make env mRNA, but a large portion must remain full length to serve as gag-pol mRNA and as genome for progeny virus.

Question 21

What are the three fates of viral RNA?

Answer 21

1. Full length RNA because genomic RNA and is used as genome for new progeny
2. Full length RNA used to make gag-pol mRNA
3. RNA splicing occurs to make env mRNA by splicing

Question 22

Why would interfering with viral splicing not be a great therapeutic target?

Answer 22

Because we have splicing too and it would also impact the ability for our cells to splice.

Question 23

Describe protein synthesis that occurs in HIV?

Answer 23

GAG initiates at the AUG start codon and ends at stop codon of gag.

Gag-pol is made from same AUG but sometimes ribosomes ignore or circumvent the Gag stop codon and continue to the end of pol ignoring the gag stop codon.

Both gag and pol are eventually cleaved by the protease (PR) domain, releasing the individual proteins.

Question 24

How is the env protein made? What must occur?

Answer 24

From spliced mRNA on the ER-bound ribosomes, and moves through the ER-golgi and is inserted into the PM.

Env gp 160 is cleaved into gp120 and gp41 by a cellular protease; this MUST occur because gp160 cannot support membrane fusion

Question 25

What occurs during virion assembly and budding?

Answer 25

Packaging requires a signal (Psi), contained in unspliced but not spliced RNA b/c splicing removed the signal.

Budding: viral gag and gag-pol recruit DNA and assemble under cell surface. gag protein interacts with env and budding occurs.

Question 26

What occurs after packaging and budding?

Answer 26

Maturation: proteolysis of gag and gagpol by PR occurs AFTER budding.

This causes protein rearrangements and the core to become more dense.

Viral particles still form and bud if proteolysis is inhibited, but the virus will not be infectious.

Question 27

Blocking maturation is the basis for what type of HIV medications?

Answer 27

HIV PR inhibitors

Question 28

What is retroviral mediated oncogenesis? How were these discovered?

Answer 28

Retroviruses were discovered as agents isolated from naturally occurring tumors in animals, that when inoculated in naive animals, would again cause tumors.

These viruses has a major impact on cancer biology.

Question 29

What are non-transforming retroviruses? How do they work?

Answer 29

Tumors take 6 mo to 1 yr to appear and do NOT transform cells in culture.

Viruses do not contain oncogenes; tumors caused by activation or inactivated of host genes.

***This is the special case where the 3’ LTR is activated, previously we said only 5’ was activated.

Question 30

Describe the tumors caused by transforming retroviruses? How fast do tumors occur?

Answer 30

Infection causes tumors within weeks and efficiently cause tissue culture cells to become transformed or be cancer-like.

Viruses harbor an “oncogene” and infection introduces the cancer causing gene causing rapid tumor onset.

Question 31

What was the first oncogene identified?

Answer 31

Src.

It was a sarcoma virus and in 1989 it was appreciated that the gene was “stolen” from the host cell.

Question 32

Why are most oncogene-containing viruses defective?

Answer 32

Because the oncogene replaces one or more viral genes and therefore wont go into mechanism.