ssRNA RT viruses

Question 1

Baltimore classification of RT ssRNA viruses

Answer 1

•RT ssRNAviruses (retroviruses) belong to Class VI, according to the Baltimore classification of viruses

Question 2

Introduction to RT dsDNAviruse

Answer 2

• Retroviruses are RNA viruses that must copy their genome into DNA in the course of transcription.
• Reverse transcription is one unique features among the classes VI and VII (dsDNA) viruses.
• Retroviruses can infect all vertebrate classes such as fish, birds, amphibians and mammals.
• Important examples of retroviruses are HIV-1 and HIV-2.
• Most retroviruses are important agents of canc

Question 3

Classification of retroviruse

Answer 3

ssRNA RT into two groups

simple and complex retroviruses

Question 4

simple retroviruses

Answer 4

Simple retroviruses: Viruses that possess only the major genes (gag, pol, env) or may have an additional gene known as an oncogene (whose expression can result in tumor cells in the host). An example is the srconcogene in the genome of Rous sarcoma virus, known to infect chickens

ssRNA RT

Question 5

complex retroviruses

Answer 5

Complex retroviruses: Possess multiple genes which perform various roles in the virus. An example is the HIV which has auxiliary genes such as, virioninfectivity factor (vif), viral protein R (vpr), viral protein U (vpu).

ssRNA RT

Question 6

How is the retrovirus virion unique

Answer 6

• The virionof a retrovirus is unique in that it has two copies of the genome.
• Thus, both RNA molecules form a dimer, by base pairing between complementary sequences.
• Incorporation of host molecules in retrovirus genome: During virionassembly and exit, retroviruses incorporates host cell RNA molecules in its genome

Question 7

tRNAin virionof Retroviruses

Answer 7

• Primer binding site (PBS): Contains sequence of virus RNA which binds with host-cell-derived tRNA.
• Binding of virus RNA to host tRNAis specific for each retrovirus

Question 8

Important proteins in the virionof Retroviruses

Answer 8

1. Nucleocapsid(NC): It coats the virus RNA, thus forms the most abundant protein in retroviruses. Other proteins are present in trace amount and mostly functions as enzymes
2. RNA-Dependent DNA polymerase (reverse transcriptase; RT).
3. DNA-dependent DNA polymerase
4. RibonucleaseH (RNaseH)
5. Integrase
6. Proteas

Question 9

The capsid of retroviruse

Answer 9

• Retrovirus capsid may have the shape of a cylinder, sphereor a coneand varies among virionspecies.
• Capsid proteins (CP): The capsid is composed of CP which surrounds the virionRNA (genome).
• Matrix(MA): A layer of protein which lies between the capsid and envelope.
• Envelope (env):is associated with two proteins which are bound non-covalently to each other.

1. Transmembrane(TM) protein:
2. glycosylated surface (SU) protein

Question 10

The genome of ssRNA retroviruses

3 genes

Answer 10

• The gene of retroviruses encodes virus proteins which are arranged in three distinct regions of the genome.
• gag(group specific antigen) encodes internal structural protein
• pol (polymerase) encodes enzymes
• Env(envelope) encodes envelope protei

Question 11

Replication in Retroviruses

steps

Answer 11

The steps in replication are: •Attachment and Entry•Reverse transcription•Transcription and Genome replication•Translation•Assembly•Exi

Question 12

attachment of ssRNA retroviruses

Answer 12

• Attachment: occurs through binding of the surface (SU) protein on the virionenvelope with host cell receptors.
• The binding results in a conformational change in the transmembraneprotein (TM) permitting a hydrophobic fusion sequence to bind the virionmembrane and a cell plasma membran

Question 13

Entry of retroviruses

Answer 13

•Cell entry: Majority of retroviruses enter host cells by fusion of virionmembrane with host cell membrane, while some gain entrance through endosome-mediated endocytosi

Question 14

Formation of reverse transcription complex in ssRNA RT viruses

Answer 14

• Formation of reverse transcription complex: Is formed upon virionentry into the cell cytoplasm which results in the loss of some proteins.
• The synthesis of both the (+) DNA and (-) DNA strands starts at the 3’-OH of a primer RNA (tRNA).
• While the primer for synthesis of (-) DNA is the tRNAbound to the genome, the primer for synthesis of (+) DNA is a polypurinetract (PPT) in the virus genom

Question 15

Figure 3 : Reverse transcription in retroviruses ssRNA

Answer 15

1. A copy of the virus genome with a tRNAbound at the PBS.
2. The reverse transcriptase begins (–) DNA synthesis at the 3′ end of the tRNA.
3. The RNaseH digests the RNA from the RNA–DNA duplex. The (–) DNA attaches atthe 3′ end of either the same RNA strand or the second copy of the genome.
4. Elongation of the (–) DNA continues, while the RNaseH degrades the template RNAfrom the 3′ end as far as the PPT.
5. Synthesis of (+) DNA begins.
6. The remaining RNA is degraded.
7. The (+) DNA detaches from the 5′ end of the (–) DNA template and attaches at the 3′end.
8. Synthesis of both DNA strands is completed.

The dsDNAresulting from the reverse transcrip

Question 16

Transport of the ssRNA RT

provirus into the nucleus

Answer 16

• Formation of pre-integration complex: Prior to entry of the provirus into the nucleus, it forms an association with some virionproteins.
• Proteins associated with the pre-integration complex are: reverse transcriptase, matrix proteinand integrase(bound to each end of the DNA).
• Transportation of the integration complex in some retroviruses (e.g. HIV) to the nucleus is mediated by microtubule complex.
• Entry of most retroviruses into the nucleus usually occur during mitosis as it requires breakdown of the nuclear envelope.
• Implication for these viruses is that productive infection can only occur during cell division.
• Note that the pre-integration complexes of HIV and related viruses can initiate productive infection at any phase of the cell cycle as they can enter an intact nucleus. Thus, do not require the cell to be in a phase of cell division prior to entry

Question 17

Figure 4: Transport of the ssRNA RT provirus into the nucleus and integration into cell DNA

Answer 17

The result of entry of pre-integration complex into the nucleus is as follows:

i. Integrasecuts the DNA of the cell chromosome in two and insert the provirus gene in between.
ii. Integration of provirus gene with cell DNA is mediated by cell DNA repair machinery.
iii. Integration could result in productive infection(immediate virus gene expression) or latent infection(delayed virus gene expression).
iv. When host cells with latent infection divide, the provirus gene is retained along with cell genome i

Question 18

Transcription and genome replication in ssRNA retroviruses

Answer 18

• Each termini of the dsDNAof the provirus has a long terminal repeats (LTRs) denoted by U3-R-U5, having a U3 sequence at one end and U5 sequence on the other end.
• Both LTRs of the provirus have identical sequences, but possess different functions as transcription in triggered in one and terminated at the othe

Question 19

Figure 6: Transcription and genome replication in retroviruses

Answer 19

• Transcription is initiated through binding of transcription factors to a promoter in upstream LTR
• Cell RNA polymerase II begins transcription at the U3-R
• Transcription proceeds into the downstream LTR and terminates at the R-U5 end which is polyadenylatedat the R region.
• Each transcript is capped and polyadenylatedat each end.
• Some transcript will serve as genome for progeny virion, while others will function as mRNA, and a portion become splice

Question 20

Translation and post-translational modification in retroviruses in the envelope

Answer 20

• Envelope: This is coded for in the envgene and translated from spliced mRNA.
• Translation of envelope proteins occurs in the rough endoplasmic reticulum, where post-translational modification (glycosylation) is also initiated.
• The translated envelope proteins are then transferred to the Golgi apparatus, where they are cleaved into SU and TM by host proteolyticenzymes.
• Both cleaved SU and TM proteins remain closely linked, undergo further glycosylation before transported to the plasma membrane

Question 21

Figure 7: Translation and post-translational

Answer 21

Spliced mRNA are transported from the nucleus to the rough endoplasmic reticulum where translation into envelope proteins occurs and glycosylation is initiated.

Envelope proteins are transported to the Golgi complex where they are cleaved into TM and SU proteins and undergo further glycosylation.

Both products are transported to the plasma membrane

Whole genome mRNA (transcribed from gag and pol genes) are translated into Gag and Gag-Pol polyproteins, for which the former is needed in large quantity than the latter

Question 22

Assembly and release of virionsin retroviruses

Answer 22

• Most retroviruses assembly their components at the inner side of the plasma membrane.
• However, some form pre-mature nucleocapsidwhich are transferred to the plasma membrane.
• Base pairing of complementary sequences between two copies of genome RNA results a dimer, whose interaction forms a ‘’kissing loop complex’’.

Question 23

Figure 7: Early-phase assembly in retroviruses:

Answer 23

Assembly of viriongene and proteins accumulate at the inner side of the plasma membrane.

A genome dimer is linked to cell-derived tRNAalong with Gag and Gag-Pol proteins.

Gag and Gag-Pol proteins become attached to the plasma membrane by the myristylgroups at their N termini

The matrix (MA) domain binds to the cytoplasmic tail of TM proteins in the membra

Question 24

Figure 8: Late-phase assembly and exit in retroviruses:Immature virionderives its envelope through budding from the plasma membrane

Answer 24

Gag and Gag-Pol polyproteinare cleaved by virus protease during and after budding of the virion.

Cleavage of Gag protein result in the capsid, the matrix and protein component of the nucleocapsid.

Cleavage of the Pol results in the virionenzymes (polymerase, intergrase, RNase, protease).

Question 25

Exit of ssRNA retroviruses and infection of new cells

Answer 25

•Progeny virionsmay initiate immediate infection of a new host cell, if it is close to the infected cell.

•Virology synapse: Connection between a virion infected cell and a susceptible cell
=> Synapse formation protects the progeny virionsfrom host immune attack.

•HIV and human T-lymphotrophicvirus 1 utilize the virology synapse as a means of infecting new cells

Question 26

Application of reverse transcriptases

Answer 26

•The role of RTs in copying RNA to DNA, even without the presence of tRNAprimers have useful application in molecular biology.

1. Production of cDNAlibraries
2. Reverse transcriptase-polymerase chain reaction (RT-PCR).

•Common sources of RTs are:

1. Moloneymurine leukemia virus
2. Avian myeloblastosisvirus

Question 27

Application of retroviruses as gene vectors

Answer 27

•Some retroviruses have been genetically modified to introduce genes of interest into the genome of different cell types.

Question 28

Application of retroviruses as gene vectors

Murine leukemia virus

Answer 28

Murine leukemia virus: has useful application as vectors for expressing genes in cell cultures and for clinical treatments of genetic disorders and cancers

Question 29

Application of retroviruses as gene vectors

Lentiviralvectors

Answer 29

Lentiviralvectors: have useful application as gene vectors in non-dividing cells and tissues. Recombinant lentivirushave been used as vectors in the treatment of patients with the

Question 30

Application of retroviruses as gene vectors

•Procedure:

Answer 30

• Procedure: Stem cells are harvested from patient and transfected with recombinant lentiviruscontaining the functional gene. Successful integration and expression of the functional gene into the patient cell genome will result in a normal immune system.
• Negative effect: The success of the use of lentivirusesin gene therapy have been hindered by the development of cancer in some patie