Final- Retroviruses

Question 1

Generals about Retroviridae

Answer 1

A very large family of animal and human viruses

Causative agents of some types of cancer, immunosuppression or

immunodeficiency diseases

May exist as stable components of the host genome

Named “retrovirus” because of an important enzyme they encode “reverse transcriptase” – David Baltimore & Howard Temin

Question 2

• Retro

Answer 2

• Retro = reverse/backward – reflects the character of the viruses to use the RNA genome to produce DNA intermediates by means of “RNA-dependent DNA polymerase” which is found in the virion of all retroviruses

Question 3

Reverse transcriptase

Answer 3

• Reverse transcriptase is an RNA-dependent DNA polymerase

Question 4

Retroviruses of veterinary importance were among the first retroviruses to be recognized e.g.,

Answer 4

• Equine infectious anaemia virus, bovine leucosis virus and jaagsiekte virus of sheep – described in 1800s

• Retroviruses of chicken – detected by Bang and Ellerman in 1908 in Denmark
• Rous sarcoma virus – detected by Rous as early as 1911 in species such as cattle,

cats, primates and mice

• Since 1980 we know that retroviruses cause human diseases too e.g., some lymphomas, leukemia and AIDS

Question 5

Characteristics of retroviruses

Answer 5

• They are enveloped viruses formed by budding from the cell

membrane
• Have a diploid genome with two copies of single stranded, positive

sense RNA

• All replicate with use of the reverse transcriptase, an enzyme encoded by the virus

Question 6

Positive sense RNA means:

Answer 6

RNA serves as mRNA from which protein translation can directly begin

Question 7

What is special about Retroviruses?

Answer 7

They integrate into the genome of the host by means of an enzyme called integrase

IMPLICATION: they may alter or acquire host genome sequences, may activate or inactivate particular host genes located near the integration site

Question 8

What is the taxonomy of Retroviridae?

Answer 8

Question 9

Morphology of retroviridae

Answer 9

Virions are spherical

Surrounded by an envelope consisting of a lipid membrane bilayer

The surface is studded by projections of envelope glycoprotein

A spherical layer of protein (matrix)

beneath the envelope

An internal capsid protein which encloses the viral RNA = nucleocapsid

• Inside the nucleocapsid - two single strand viral RNA, reverse transcriptase, integrase and protease

Question 10

Virion properties

Answer 10

• Diameter 80-100 nm
• Each RNA genome is between 7 and 11 kb and has a 3’

polyadenylated tail and a 5’ cap
• Because virions are enveloped, they are easily inactivated by lipid

solvents or detergents and by heating
• Virions are resistant to ultraviolet or X-rays

Question 11

What is required for replication of these viruses?

Answer 11

• At least 30 molecules of reverse transcriptase (RT) are included in the virion
• RT is required for replication
• RT serves as:
• RNA-dependent DNA polymerase
• DNA-dependent DNA polymerase • Rnase
• Virion has at least 3-4 important genes

Question 12

What are the different genes that in the viruses?

Answer 12

gag, pol, env,, pro

Question 13

The gag gene encodes

Answer 13

• The gag gene encodes the viruses core proteins
• Capsid (CA)
• Nucleocapsid (NC)
• Matrix (MA)

Question 14

The pol gene encodes:

Answer 14

The pol gene encodes:
• Reverse transcriptase (RT)

• Integrase (IN)

Question 15

The env gene encodes:

Answer 15

The env gene encodes:
• Virion envelope proteins, surface (SU) and transmembrane (TM)

Question 16

The pro gene encodes

Answer 16

The pro gene encodes the protease

Question 17

In lentiviruses

Answer 17

• the tat gene encodes a transactivator that enhances the efficiency of

cellular RNA polymerase
• the rev gene encodes a protein that facilitates transport of non-

spliced viral RNA from the nucleus to the cytoplasm

• the nef gene encodes Nef protein that:
• is essential for replication in target hosts but not in cell culture in vitro
• may downregulate expression of the CD4 molecule or alter the activation status of target cells
• the vif gene encodes a protein Vif which enhances replication in lymphocytes

Question 18

Alpharetroviruses - characteristics

Answer 18

• Referred to as simple retroviruses
• Have a type C virion morphology
• Represented by avian leucosis and sarcoma virus (ALSV)
• The genome contains gag, pro, pol and env genes
• The pro gene is at the 3’ end of gag in the same reading frame • Mostly endogenous and exogenous viruses of chicken

Question 19

Exogenous retroviruses

Answer 19

Exogenous retroviruses are those viruses transmitted horizontally and are rarely transmitted via in-utero or germline infection

Question 20

Endogenous retroviruses

Answer 20

Endogenous retroviruses (retroviral elements) are those viruses included in the genome of most if not all animals

• Are transmitted vertically as part of the host genome • Are passed on from generation to generation

Question 21

Betaretroviruses

Answer 21

• Simple retroviruses

Have type B morphology with round eccentric core or type D with cylindrical core

Exemplified by mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (M-PMV)

Virions are assembled in the cytoplasm via A type intermediate

The genomes contain gag, pro, pol and env genes

gag, pro and pol are all in different reading frames

MMTV additionally has the sag gene which encodes the superantigen

The genus contains both endogenous and exogenous retroviruses

Question 22

Gammaretroviruses

Answer 22

Simple retroviruses with a C type morphology

Has the largest number of members e.g., murine leukemia virus, feline leukemia virus etc.

The genome contains gag, pro, pol and env

The gag, pro, pol are in the same reading frame

They contain both endogenous and exogenous retroviruses of mammals and reptiles

Question 23

Deltaretroviruses

Answer 23

• Complex retroviruses with a C-type morphology
• The famous examples are: human T-lymphotropic virus (HTLV), bovine leukemia virus
• The genome contains gag, pro, pol, env and regulatory genes rex and tax
• The gag, prol and pol are presented in 3 different reading frames
• Expression of Gag-Pro-Pol requires 2 successive frame shifts
• Rex and Tax are expressed from an alternatively spliced mRNA • Rex and Tax control synthesis and processing of viral RNA
• There are no known endogenous deltaretroviruses
• Exogenous retroviruses are found only in a few mammals

Question 24

Epsilonretroviruses

Answer 24

Complex retroviruses with a type C morphology

Prototype is the Walleye dermal sarcoma virus

Contain gag, pro, pol and env with gag, pro and pol in the same reading

frame

Additionally, 3 more genes termed ORF A, B, and C are present

The ORFa is a viral homologue of the host cyclin D and may regulate host cell cycle

Only endogenous retroviruses are found in fish and reptiles

Question 25

Lentiviruses

Answer 25

* Complex viruses with a cylindrical or conical shaped core * The most important members are the human immunodeficiency virus (HIV) and non-primate viruses such as caprine arthritis-encephalitis virus and visna virus * The genome contains gag, pro, pol and env * gag is in a separate reading frame while pro and pol are in the same reading frame * A single frame shift is used to express Gag-Pro-Pol

Question 26

What are the acessory genes in Lentiviruses?

Answer 26

* Accessory genes such as vif, vpr, vpo, tat, rev and nef are present * Accessory genes control transcription, RNA processing, virion assembly, host gene expression and other replication functions * Exogenous retroviruses are found in many mammals

Question 27

Lentiviruses have extra genes

Answer 27

In lentiviruses The tat gene encodes a transactivator that enhances the efficiency of the cellular RNA polymerase The rev gene encodes a protein that facilitates transport of non-spliced viral RNA from the nucleus to the cytoplasm The nef gene encodes NEF protein that: is essential for replication in target hosts but not in cell culture in vitro may downregulate expression of the CD4 molecule or alter the activation status of target cells The vif gene encodes a protein VIF which enhances replication in lymphocytes

Question 28

Spumaviruses

Answer 28

* Complex viruses with a unique virion morphology containing spikes on the surface, a central but uncondensed core * Exemplified by human foamy virus Virions are assembled in the cytoplasm and budded into endoplasmic reticulum (ER) and plasma membrane The genome contains gag, pro, pol and env Accessory genes, tas/bel-1 and bet are present The tas gene encodes a transcriptional activator Exogenous viruses are found in diverse mammals

Question 29

Replication of retroviruses

Answer 29

Receptor binding and membrane fusion Internalization and uncoating Reverse transcription of the RNA genome to form linear dsDNA Nuclear entry of DNA Integration of linear DNA into the host genome to form a provirus Transcription of the provirus to form viral RNA Splicing and nuclear export of RNA to the cytoplasm Translation of the RNA to form precursor proteins Assembly of virions and packaging of viral RNA genome 10. Budding and release of the virions 11. Proteolytic processing of the precursors and maturation of the virions

Question 30

Receptors - binding

Answer 30

To enter a cell and initiate infection, retroviruses require interaction between a cell surface molecule (receptor) and the envelope proteins on the virion surface

Question 31

Alpharetroviruses Receptor binding

Answer 31

a membrane anchored glycoprotein with a sequence similar to ligand-binding repeat of the low-density lipoprotein receptor (LDLR)

Question 32

Betaretroviruses receptor binding

Answer 32

(MMTV) transferrin receptor tfr-1

Question 33

Gammaretroviruses receptor binding

Answer 33

Gammaretroviruses – several receptors, mCat-1 for mouse viruses; GLVR1 for cat, dog and primate viruses; FeLIX and Pit for FeLV

Question 34

Deltaretroviruses Receptor binding

Answer 34

a protein similar to AP-3 complex for BLV

Question 35

Lentiviruses receptor binding

Answer 35

Lentiviruses – CD4 on T helper cells, also expressed on macrophages and dendritic cells; CCR5 and CXCR4 expressed on lymphoid cells

Question 36

Penetration and uncoating

Answer 36

Upon binding to the receptor the virion fuses with the host cell membrane Mostly, entry is pH-independent (no endosomal acidification is required). Some retrovirus may require endosomal passage. Entry may require major rearrangements in the virion envelope proteins e.g., exchange of sulphide bonds between TM and SU The virion core is released into the cytoplasm of the infected cell Uncoating is not well studied

Question 37

Reverse transcription

Answer 37

• A dsDNA copy of the retroviral sRNA is made by the reverse transcriptase associated with virion In this process, about 300-1300 bp are added to each end of the genomic RNA molecule called LTR Host cell-derived transfer RNA (tRNA) (specific for each retrovirus) binds viral 5’ LTR sequence and primes the RT reaction During the copying of cDNA from the viral RNA, the RNase H degrades the RNA leaving only the sDNA * The sDNA then hybridizes with the genomic viral RNA to complete synthesis of the first strand followed by dsDNA * Linear dsDNA intermediate is then transported to the nucleus

Question 38

Integration

Answer 38

Takes place in the nucleus • Most retroviruses (except lentiviruses) **require _cell_** **_division_ in order to integrate into the host genome** * First the **3’ prime ends of viral dsDNA are processed by the integrase (IN)** to prepare for insertion * The strand is then transferred into the host DNA

Question 39

Is integration site specific?

Answer 39

**_Integration is not site specific,_** however sites such as **DNA bendings,** open chromosomal structures that are **transcriptionally active are preferred**

Question 40

provirus

Answer 40

After integration into the host cell chromosome has completed, the viral DNA is now called “**a provirus”** Basically proviruses are **templates for transcription** • The **provirus** may remain **latent** (“silent”, i.e., transcriptionally innate) or may be transcriptionally active

Question 41

Transcription

Answer 41

• Transcription is performed by **_host_ cellular RNA polymerase** to generate viral RNA **• the upstream LTR directs transcription** • Once the viral RNA is produced it is spliced and **transported** to the cytoplasm, **spliced or non-spliced**

Question 42

There are _two pools of viral mRNA_ found in the cytoplasm of an infected cell

Answer 42

* _Full-length genomic RNA t_hat is later packaged into virions as viral RNA * _Spliced mRNA_ used to express structural proteins such as **Gag, Pol and Env**

Question 43

In cells infected with retroviruses, viral protein synthesis occurs in the

Answer 43

In cells infected with retroviruses, **_viral protein synthesis occurs in the cytoplasm and is regulated similarly to host proteins_**

Question 44

what are the spliced and non-spliced RNA are used for?

Answer 44

* Spliced\* forms mRNA are used to translate envelop and accessory proteins * Full-length, non-spliced is packaged into the virion

Question 45

Splicing

Answer 45

Removal of introns from pre-messenger RNA and joining of exons to form mRNA

Question 46

Assembly and release of virions

Answer 46

* _Virion assembly_ begins once translation of proteins is finished leading to _budding_ of the virion from the cell membrane * Almost all retroviruses replicate in dividing cells * _No overt pathology_ or change in cell metabolism is observed _• **Lentivirus** cause cell death by formation of cell syncytium and apoptosis_

Question 47

Depending on the presence of the key transforming genes in the viral genome, infection with retrovirus may lead to

Answer 47

• Depending on the presence of the key transforming genes in the viral genome, infection with retrovirus may lead to * Acute transformation * Chronic transformation

Question 48

• Retroviruses that can lead to acute transformation

Answer 48

• Retroviruses that can lead to acute transformation contain viral oncogenes (v-onc)

Question 49

Retroviruses that cause chronic transformation do so by

Answer 49

Retroviruses that cause chronic transformation do so by insertion/mutation through random integration into the genome of the host

Question 50

Cellular oncogenes are required for

Answer 50

**_Cellular oncogenes are required for normal cell growth and differentiation_**

Question 51

How do retroviruses cause onocogeneisis?

Answer 51

Retroviruses acquire cellular oncogenes (c-onc) during replication These acquired c-onc become viral oncogenes (v-onc) Viral oncogenes undergo mutations which usually removes the regulatory mechanisms

Question 52

What do viral oncogenese lead to?

Answer 52

• Such a viral oncogene may lead to transformation of infected cells by acting as _growth factors, receptors, intracellular signal transducers or intranuclear transcription factors_