RNA Viruses

 

I.  General Characteristics

            -Three subtypes

                        a. (+) Strand

                        b. (-) Strand

                        c. Double Strand

-The genome of all RNA viruses except retroviruses encode an RNA-

dependent RNA polymerase.

-Transcription of viral genomic RNA and mRNA are not inhibited by

Actinomycin D, which inhibits cellular DNA-directed RNA synthesis.

            -RNA-directed RNA synthesis:

a. RNA synthesis initiates and terminates at specific sites in the template

                        b. Catalyzed by virus-encoded polymerases

                        c. May require viral and/or host accessory proteins

d. Most RNA-dependent RNA polymerases initiate synthesis denovo but some require a primer with a free 3’OH end complementary to the template strand

                        e. RNA primers may be protein linked and may contain a 5’ cap

                        f. Elongation is in a 5’ to 3’ direction

            -RNA-dependent RNA polymerases

a. The active site of most (+) strand, double strand and segmented (-) strand viruses have the motif Gly-Asp-Asp

b. The active site of most (-) strand non-segmented viruses have the motif Gly-Asp-Asn

c. Structure is analogous to a right hand consisting of a palm (active site), fingers and thumb.

            -RNA molecules contain secondary structures important for Synthesis,

Translation, and Assembly

                        a. Stem loop regions

                        b. Hairpin loops

                        c. Bulge loops

                        d. Interior loops

                        e. Multibranched loops

                        f. Psuedoknot

            -Accessory Proteins in RNA-dependent RNA synthesis

                        a. Viral proteins

                                    -Viral proteins may have various roles:

                                                i. Direct the polymerase to the correct intracellular site

                                                ii. Targets to the correct initiation site on RNA

templates

                                                iii. RNA helicases

                                                            -Unwind genomes of double stranded RNA

viruses

                                                            -Unwind secondary structures in template

RNAs

                                                            -Prevent base-pairing between template and

nacent strands.

                        b. Cellular proteins

                                    -Viruses may require different cellular proteins

            -Initiation mechanisms of RNA synthesis

a. Most RNA-dependent polymerases do not require a primer

except:           

                                    i. Picornoviuses

                                    ii. Influenza viruses

                                    iii. Bunyaviruses

                        b. Two types of Priming Mechanisms

                                    i. Protein Priming

                                                -Picornoviruses

                                                -Primer is a genome linked protein

                                    ii. Priming by capped RNA fragments

                                                -Influenza viruses and Bunyaviruses

                                                -Capped RNA primers acquired from cleavage of host

cell RNA polymerase II transcripts: Cap

Snatching

                                                -A cap-dependent endonuclease that is part of the

RNA polymerase creates 10 to 13 nucleotide

long capped primers that serve as primers for

initiation of viral mRNA synthesis.

                                                -Inhibited by the fungal toxin a-amantinin

            -Conformational Changes and Regulation of RNA synthesis

                        a. Alterations in viral RNA polymerases may influence template

recognition and initiation:

                                    i. Conformational changes

                                    ii. Proteolytic changes

            -Synthesis of (+) strand and (-) strand is not balanced

                        a. The efficiency of initiation of RNA synthesis at the 3’ end of the

(+) and (-) strands may be different

                        b. The polymerase that synthesis one type of strand may be limited

in its expression during infection

            -Most viral mRNAs contain a terminal poly-A tail

            -Mechanisms for switching from mRNA synthesis to genome RNA

synthesis

                        a. Picornovirus mRNA is identical to genome RNA.

                        b. Different RNA polymerases for mRNA synthesis and genome

replication

                        c. Suppression of intergenic stop-start reactions by nucleocapsid

proteins

                        d. Suppression of termination by Stem-loop structures

                        e. Different templates used for mRNA synthesis and Genome

replication

                        f. Suppression of polyadenylation

            -Origins of Diversity

                        a. Misincorporation of nucleotides

                                    -RNA-dependent RNA polymerases can’t proofread

                        b. Segment Reassortment

                        c. RNA recombination

                                    i. Base-pair dependent

                                    ii. Base-pair independent

                                    iii. Base-pair assisted

II. Positive strand RNA viruses

            -Virions lack a viral RNA polymerase

            -The deproteinized RNA molecules of these viruses are infectious

and can be translated in cells to produce viral proteins

            -Most lack an envelope

            -Genomes are not coated with viral proteins in the virion

            -RNA synthesis takes place on internal membranes in the cytoplasm

                        i. May allow concentration of replication components

                        ii. May allow sufficient packaging of progeny RNA into virions

                        iii. May provide lipid components or support

 

III. Negative Strand RNA viruses

            -Virions must contain and RNA polymerase

            -Incoming RNA is not infectious and cannot be copied or translated by

cellular proteins

            -Most are enveloped

            -The genomes are organized into nucleocapsids

                        a.RNA-dependent RNA polymerase and accessory proteins are

bound to genomic RNA at regular intervals

                        b. Resistant to RNase

                        c. RNA polymerase only copies RNA when it is in the nucleocapsid

and not when they are naked RNA

                        d. Nucleoproteins are single-stranded RNA binding proteins

                                    -Keep RNA single stranded

                                    -Prevent base pairing between template and product

            -RNA synthesis takes place in nucleocapsids in the cytoplasm or nucleus

           

IV. Double Strand RNA viruses

-Virions must contain and RNA polymerase

            -Incoming RNA is not infectious and cannot be copied or translated by

cellular proteins

            -Most are enveloped

            -RNA synthesis takes place in subviral particles in the cytoplasm

 

V.  Examples of RNA viruses

 

 

Orthomyxoviruses

A.     Genus

a.      Influenza A virus

b.      Influenza B virus

c.      Influenza C virus

d.      Thogotovirus (Thogoto virus)

e.      Isavirus (Infectious salmon anemia virus)

B.     Example: Influenza A virus

a.      Genome Organization

                                                                          i.      (-) strand segmented RNA genome

                                                                        ii.      Eight segments encoding 10 proteins

b.   Enveloped

c.       Functions of viral proteins

1.   Hemagglutinin

1.      Encoded by RNA segment 4

2.      Membrane glycoprotein

3.      Trimer of disulfide linked HA1 and HA2 molecules

4.      HA0 is cleaved at basic residues by proteases located in the trans golgi into HA1 and HA2 subunits

5.      Mediate binding of the virion to host cells by binding sialic acids attached to galactose with an a(2,6) linkage (human strains) or an a(2,3) linkage (avian strains)

6.      The fusion peptide of HA (n-terminal regions of HA2) mediates fusion of viral and endosomal membranes by undergoing a conformational change at pH 5

7.      Its cytolasmic domain binds the M1 protein which is attached to viral RNA during assembly

8.      Mutates rapidly

9.      Influenza vaccines strive to produce antibodies against Hemagglutinin to neutralize the virus by inhibiting cellular attachment

                                                                                                ii.      Neuramindase

1.      Encoded by RNA segment 6

2.      Membrane glycoprotein

3.      Cleaves glycoside linkages of sialic acid

a.      Inhibits aggregation of HA which are modified with sialic acid in the golgi apparatus

b.      Inhibits binding of HA to sialic acid residues on host cells as virions are released from infected cells

4.      Its cytomplasmic domain binds the M1 protein which is attached to viral RNA during assembly

5.      Mutates rapidly

6.      Influenza vaccines strive to produce antibodies against NA

                                                                                              iii.      Nucleoprotein

1.      Encoded by RNA segment 5

2.      Regulates the switch from viral mRNA to full length (+) strand synthesis by binding to the nacent (+) strand and blocks poly A addition

3.      Contains a nuclear localization signal which allows its import into the host nucleus by cellular import proteins

4.      Facilitate nuclear localization of viral RNA after it is released into the cytoplasm

5.      M1 protein binds to both NP and cytoplasmic face of HA and NA to guide vRNPs to the plasma membrane for assembly

                                                                                               iv.      Non-structural protein

1.      Encoded by RNA segment 8

2.      Inhibits cellular mRNA polyadenylation

3.      Inhibits splicing of cellular pre-mRNAs

4.      Has a N-terminal RNA binding domain

5.      Has a C-terminal domain that binds Cpsf and PabII involved in polyadenylation

6.      Cytoplasmic accumulation of mRNA may facilitate cap-snatching

7.      Sequesters dsRNA that could bind and activate Pkr (Pkr phosphorylates eIF2a which shuts down translation)

8.      Inhibits IFN synthesis

                                                                                                 v.      Matrix Protein (M1)

1.      Encoded by RNA segment 7

2.      Bind RNP and cytoplasmic tails of HA and NA

3.      May undergo a conformational change before membrane fusion facilitated by a low pH created by M2 allowing it to release vRNPs so they can travel to the nucleus

4.      It’s release is inhbited by the drug amantadine

5.      By binding to genomic RNPs in the nucleus

a.      Inhibits RNA synthesis

b.      Promotes nuclear export of RNA

6.  Has an intrinsic propensity to associate with the plasma membrane

                                                                                               vi.      NEP

1.      Encoded on RNA segment 8 by splicing

2.      Contains a C-terminal M1 protein binding domain

3.      Contains a N-terminal nuclear export signal

4.      Directs export of vRNPs out of the nucleus

                                                                                             vii.      Ion Channel (M2)

1.      Encoded on RNA segment 7 by splicing

2.      The 5’ splice site for its mRNA is suboptimal and requires binding of cellular protein sf2 to a enhancer in the 3’ exon of M1 mRNA.  This interaction requires binding of P proteins with the 5’ end of M1 mRNA

3.      Forms a homotetramer in the virion envelope

4.      Activated by the low pH of the endosome before membrane fusion occurs

5.      Allows protons to enter the virion particle which may cause a conformational change in M1 that causes its dissociation from vRNPs

6.      Inhibited by amantadine

7.      Increases the pH of normally acidid compartments of the trans-golgi keeping HA in a fusion-incompetent state.

                                                                                           viii.      PA

1.      Encoded on RNA segment 2

2.      Part of the polymerase complex

3.      Important for replication but how its initiation properties are regulated is uncertain

                                                                                               ix.      PB1

1.      Encoded on RNA segment 1

2.      Part of the polymerase complex

3.      Binds to the 5’ terminal sequence of genomic RNA

4.      Binds to a 3’ terminal sequence of genomic RNA activating its ability to cleave capped RNA

5.      Cleaves capped cellular RNA

6.      Catalyzes each nucleotide addition

7.      Binds to a specific 5’ sequence on viral mRNA inhibiting it’s cap cleavage

                                                                                                 x.      PB2

1.      Encoded on RNA segment 3

2.      Part of the polymerase complex

3.      Binding of PB1 to its RNA sequence induces a conformational change allowing it to bind the 5’cap of cellular RNA

d.      Single-cell reproductive Cycle

a.      HA binds a sialic acid-containing cellular surface protein or lipid

b.      The virion enters the cell via receptor-mediated endocytosis

c.      Acidification of the endosome induces a conformational change in M2 promoting proton influx

d.      M1 undergoes a conformational change facilitating its release from viral RNPs

e.      HA undergoes a conformational change that exposes its fusion peptide, facilitating viral and endosomal membrane fusion

f.        Viral nucleocapsids are released into the cytoplasm

g.      Viral nucleocapsids containing the segmented genome, NP and Polymerase proteins are transported to the nucleus via nuclear localization signals on NP

h.      The (-) RNA genome is used to synthesize viral mRNA via the viral polymerase complex which uses 5’ caps of cellular RNA as a template

i.        Viral mRNAs (spliced and unspliced) are transported to the cytoplasm by normal cellular mechanisms through the nuclear pore complex

j.         Viral mRNAs of HA, NA and M2 are translated on ribosomes on the ER and enter the secretory pathway  where they are modified by glycosylation.

k.      All other mRNAs are encoded on ribosomes in the cytoplasm

l.         P proteins and NP are transported to the nucleus to facilitate synthesis of (+) strand RNA and genomic RNA

m.     M1, NEP and NS1 proteins are transported to the nucleus and shut down viral mRNA production and facilitate nuclear export of progeny nucleocapsids to the cytoplasm. 

n.      M1 association with NA and HA in the plasma membrane facilitates virion assembly

o.      The virion buds of from the host cell

p.      NA cleaves sialic acids to prevent HA binding during budding

e.      Disease: Influenza

a.      Symptoms

                                                                                                  i.      Acute febrile respiratory tract infection

                                                                                                ii.      Rapid onset of fever, malaise, sore throat, cough

b.      Transmission

                                                                                                  i.      Inhalation of small aerosol droplets

                                                                                                ii.      Most common in winter when people are indoors

c.      Mechanism

                                                                                                  i.      Infects upper and lower respiratory tract

                                                                                                ii.      Systemic symptoms caused by a cytokine response

                                                                                              iii.      Neutralizing antibodies to HA and NA are produced

                                                                                               iv.      Recovery depends on a cell-mediated and interferon response

                                                                                                 v.      May get a secondary bacterial infection due to loss of natural epithelial borders

d.  Treatment Strategies

            1.  Killed vaccine containing antigens of two influenza

A viruses (H1N1 and H3N2) and a influenza B

virus

            2.  Live attenuated influenza A and influenza B

vaccine (FluMist)

            3.  Antiviral drugs

                        1. Amantadine

                                    -Inhibits the M2 ion channel

                                    -Results in retention of M1 to vRNPs

                        2.  Rimantadine

                        3.  Zanamivir

                                    -inhibits NA

                                    -delivered via inhalation

                        4.  Oseltamivir

                                    -inhibits NA

                                    -given orally

e.  Detection Methods

            1.  Cell culture of nasal swab

            2.  Immunofluorescence

            3.  Z-stat test

 

Picornaviruses

A.     Genus

a.      Enterovirus (poliovirus)

b.      Rhinovirus (Human Rhinovirus A)

c.      Cardiovirus (Encephalomyocarditis virus)

d.      Aphthovirus (Foot and mouth disease virus)

e.      Hepatovirus (Hepatitis A)

f.        Parechovirus (Human Parechovirus)

g.      Erbovirus (Equine rhinitis B virus)

h.      Kobuvirus (Aichi virus)

i.        Teschovirus (Porcine teschovirus 1)

B.     Example:  Poliovirus

a.      Genome Organization

                                                                          i.      (+) Strand RNA genome

                                                                        ii.      Vpg protein is covalently attached to the 5’ end

                                                                      iii.      Its RNA is uncapped and translated by IRES

b.      Non-enveloped

c.      Function of viral proteins

                                                                          i.      VP1

1.      Part of the 5s structural unit

2.      Upon virion binding to the Pvr receptor at temperatures higher than 33 degrees Celsius, the virion undergoes a receptor mediated conformational change that exposes its hydrophobic N-terminus

3.      Its N-terminus inserts into the host cell membrane forming a pore through which viral RNA is released from the capsid into the cytosol

                                                                        ii.      VP2

1.      Part of the 5s structural unit

2.      Part of VP0 with VP4 until viral RNA has been encapsidated ant VP0 is cleaved by 3CDpro

3.      Myristoylated

                                                                      iii.      VP3

1.      Part of the 5s structural unit

                                                                       iv.      VP4

1.      Part of the 5s structural unit

2.      Part of VP0 with VP2 until viral RNA has been encapsidated ant VP0 is cleaved by 3CDpro

3.      Myristolated N-terminus interacts with VP3

4.      Missing from the capsid once it has undergone a conformational change upon receptor binding

5.      Required for an early stage of cell entry

                                                                         v.      2Apro

1.      A protease

2.      Cleaves the polyprotein precursor between P1 (capsid) and P23 (protease and RNA synthesis)

3.      Cleaves eIF4GI to inhibit translation of cellular mRNA.  The c-terminus of eIF4G is still needed for recruitment of the 40s ribosome to IRES

                                                                       vi.      2B

1.      Inhibits membrane transport from the ER to the plasma membrane

2.      Synthesis of 2BC induces membrane vesicles by interacting with the CopII complex and may prevent fusion with golgi by stabilizing the CopII complex.  2BC, 2B and 3A can all inhibit membrane transport from the ER but only 2BC results in the typical polio-induced membrane vesicles being produced

                                                                     vii.      2C

1.      Helps bring RNA polymerase to membranes

2.      Anchors viral RNA to membranes during replication

                                                                   viii.      3AB

1.      Helps bring RNA polymerase to membranes (Binds 3Dpol)

2.      Anchors the viral protein primer (Vpg) for RNA synthesis to cell membranes

                                                                       ix.      3CD

1.      Precursor of 3Cpro and 3Dpol

2.      The 3C portion contains the RNA binding domain

3.      During (-) strand synthesis, binds the 5’ cloverleaf structure of (+) strand along with poly (rC) binding protein 2.  Interacts with the poly A binding protein (PAbp) producing a circular genome.  Cleaves membrand bound 3AB to produce Vpg and 3A.  Binds to the internal cre site to facilitate Vpg-pUpU synthesis which is then transferred to the 3’ end.

4.      During (+) strand synthesis it binds to the cloverleaf in the (+) strand to separate the two strands and cleaves 3AB into Vpg and 3A and cleaves itself to form 3Cpro and 3Dpol

                                                                         x.      3Cpro

1.      Cleaves VP0, VP1, and VP3 from P1

2.      Cleaves 2A, 2B, and 2C from P2

3.      Cleaves 3AB and 3CD from P3

4.      Cleaves Vpg and 3A from 3AB

5.      Cleaves 3Cpro and 3Dpol from 3CD

6.      Cleaves the Tbp subunit of TFIIIb inhibitin RNA polymerase II

                                                                       xi.      3Dpol

1.      The RNA-dependent RNA polymerase

d.      Single-cell reproductive cycle

                                                                          i.      Virion binds to cellular Pvr receptor stimulating rearrangement

                                                                        ii.      The hydrophobic N-terminus of VP1 is exposed and inserts into the plasma membrane of the host cell creating a pore

                                                                      iii.      Viral RNA is released into the cell cytoplasm through the pore

                                                                       iv.      The 5’ attached Vpg protein is removed from viral RNA

                                                                         v.      The 40s ribosome associates with the IRES of the viral RNA via eIF4G, eIF4A, and eIF3 interactions and binds near or at the AUG start codon

                                                                       vi.      RNA is translated into a polyprotein that is cleaved by 2A and 3C

                                                                     vii.      (+) strand RNA is brought to cellular vesicle membranes to initiate (-) strand synthesis

1.      2BC, 2B and 3A all inhibit membrane transport from the ER and 2BC induced typical polio-induced membrane vesicles by interacting with CopII

2.      2C anchors viral RNA to membranes vesicles

3.      3AB is anchored to the vesicle and serves as a precursor for Vpg and can also binds 3Dpol to recruit it to the membrane

4.      poly(rC)-binding protein 2 (PCbp2) and 3CDpro bind the 5’cloverleaf in the (+) strand of RNA

5.      3CDpro cleaves 3AB into Vpg and 3A

6.      3CDpro binds to the poly A binding protein to create a circular genome

7.      The internal cre sequence binds 3CDpro 3Dpol and Vpg

8.      Vpg-pUpU is synthesized by 3Dpol using the cre AAACA sequence as a template

9.      The complex is transferred to the 3’end and 3Dpol uses Vpg-pUpU as a template for (-) strand synthesis

                                                                   viii.      (-) RNA strand is used to produce more (+) strand RNA for translation and packaging into virions

1.      The replicative form (-) strand is separated from the parent strand by 2C which binds to the cloverleaf in the (-) strand and PCbp2, 3CDpro, and 3AB which bind to the cloverleaf in the (+) strand

2.      3CDpro cleaves 3AB into Vpg and 3A

3.      3CDpro cleaves itself into 3Cpro and 3Dpol

4.      VPg-pUpU is synthesized by 3Dpol using the 3’ end of the (-) strand as a template

5.      3Dpol synthesizes the (+) strand

                                                                       ix.       Virion is assembled by formation of 5S structural units (VP0, VP1 and VP3) that assemble into 14S pentamers.

                                                                         x.      14S pentamers  associate with (+) strand viral RNA to form a 150S non-infectious virion stabilized by protein-protein interactions and interactions mediated by myristate chains of VP0

                                                                       xi.      An infectious 150S virion is formed by  cleavage of VP0 into VP2 and VP4.

                                                                     xii.      Release from host cell by lysis (mediated by 3A induced increased in membrane permability) or non-destructive apical release as seen in GI epithelial cells

 

e.      Disease: Polio

                                                                          i.      Symptoms

1.      Paralysis

2.      Enchephalitis and meningitis

3.      Respiratory tract infections

4.      Undifferentiated fever

5.      Disease in immunodeficient patients

                                                                        ii.      Transmission

1.      Fecal-oral route

2.      Polio is nearly eradicated

                                                                      iii.      Mechanism

1.      Enters oropharyngeal or intestinal mucosa and replicates

2.      Secretory IgA can prevent infection

3.      Travels to the lymph note

4.      Spread by Viremia to the brain and meninges

5.      Virus shed in feces

                                                                       iv.      Treatment Strategies

1.      Live oral or inactivated vaccines

                                                                         v.      Detection

Reoviruses

A.     Genus

a.      Orthoreovirus (Mammalian orthoreovirus)

b.      Orbivirus (Bluetongue Virus)

c.      Rotavirus (Rotavirus A)

d.      Coltivirus (Colorado tick fever virus)

e.      Aquareovirus (Aquareovirus A)

f.        Cypovirus (Cypovirus 1)

g.      Fijivirus (Fijivirus 1)

h.      Phytoreovirus (Rice dwarf virus)

i.        Oryzavirus (Rice ragged stunt virus)

B.     Example:  Mammalian Orthoreovirus

a.      Genome Organization

                                                                          i.      Double stranded RNA

                                                                        ii.      10 dsRNA segments

                                                                      iii.      Viral RNA polymerase and other enzymes needed for mRNA synthesis are packaged in the particle

b.      Non-enveloped and Icosahedral

c.      Function of Viral Proteins

                                                                          i.      l1

1.      Makes up the dense core shell

                                                                        ii.      l2

1.      5” capping enzyme

2.      Guanylyltransferase

3.      N-methyltransferase

                                                                      iii.      l3

                                                                       iv.      m1

1.      Makes up the outer capsid

2.      Mediates interaction of ISVP with membranes

3.      Present in the virion as two cleaved fragements m1C and m1N.  Cleaved by lysosomal proteases.  They function in membrane penetration

                                                                         v.      m2

                                                                       vi.      mNS and mNSC

                                                                     vii.      s1 and s1s

1.      Attaches to the cell receptor (sialic acids)

                                                                   viii.      s2

1.      Makes up the dense core shell

                                                                       ix.      sNS

                                                                         x.      s3

1.      Makes up the outer capsid

2.      Released by proteolysis in the ISVP

3.      Sequesters dsRNA to inhibit Pkr activation

d.      Single-cell reproductive cycle

                                                                          i.      The virion attaches to the host cell via s1 binding sialic acid on the host cell

                                                                        ii.      The virion enters by receptor-mediated endocytosis

                                                                      iii.      Proteolysis in the late endosome by lysosomal proteases of s3 and m1 creates the ISVP (infectious subviral particle)

                                                                       iv.      m1N and m1C mediate membrane penetration

                                                                         v.      The core is formed by releases of 12 s1 and 600 m1 subunits

                                                                       vi.      Synthesis of 10 capped viral mRNAs begins in the core

                                                                     vii.      l2 facilitates 5’ capping of mRNA which lacks a poly-A tail

                                                                   viii.      mRNAs are translated and associate with newly synthesized viral proteins to form RNase sensitive subviral particles

                                                                       ix.      mRNA also serves as a template for (-) strand RNA synthesis leading to the production of dsRNA  containing RNase-insensitive subviral particles

                                                                         x.      mRNAs in subviral particles facilitate further protein synthesis

                                                                       xi.      Preformed complexes of outer capsid proteins are added to subviral particles

                                                                     xii.      Mature virus particles exit the cell by lysis

e.      Disease

                                                                          i.      Symptoms

1.      Mild upper respiratory tract disease

2.      Gastroenteritis

3.      Biliary atresia

                                                                        ii.      Transmission

1.      Fecal-Oral route

                                                                      iii.      Mechanism

1.      Ingestion of contaminated food or water

2.      Large quantities of virions released in diarrhea

3.      Immunity to infection depends on IgA in the gut

                                                                       iv.      Treatment Strategies

1.      None

                                                                         v.      Detection

Rhabdoviruses

A.     Genus

a.      Vesiculovirus (vesicular stomatitis virus Indiana)

b.      Lyssavirus (Rabies Virus)

c.      Ephemerovirus (Bovine Ephemeral fever virus)

d.      Novirhabdovirus (infectious hematopoietic necrosis virus)

e.      Cytorhabdovirus (Lettuce nectrotic yellows virus)

f.        Potato yellow dwarf virus

B.     Example:  Vesicular Stomatitis Virus

a.      Genome Organization

                                                                          i.      (-)Strand RNA

                                                                        ii.      Non-segmented

b.      Enveloped

c.      Function of Viral Proteins

                                                                          i.      N (Nuceloprotein)

1.      Complexes with P at a 1:1 molar ratio

2.      Aggregates when synthesized alone

3.      NP-Complexes suppress mRNA synthesis by binding to leader RNA causing anti-termination

4.      Additional N proteins bind to the (+) RNA as it elongates and 7 A bases in the intergenic region.  Prevents the  A bases from slipping back on genomic RNA and blocks reiterative coping of the 7 U bases by L-P polymerase

5.      As genomic RNA is synthesized, 10 N molecules and 5 P molecules bind 90 nts of RNA to form oligomers facilitating ribonucleotide formation

                                                                        ii.      P (RNA polymerase)

1.      Forms a complex with L (L-P complex) to form an RNA polymerase that synthesizes mRNA

2.      Forms a complez with N to form a (L-PN) complex that synthesizes viral genomic RNA

3.      NP complexes suppress mRNA synthesis by binding to leader RNA causing anti-termination

4.      Forms oligomers with N proteins and genomic viral RNA to form ribonucelotides

5.      Bind the RNA genome in the virion

                                                                      iii.      M (Matrix Protein)

1.      Binds to the internal domain of envelope glycoprotein G and to Ribonucleoproteins (N-terminus) aiding in packaging of viral RNPs

2.      Inhibits transcription by host RNA polymerase II

                                                                       iv.      G (Glycoprotein)

1.      Has a palmitate link

2.      Associates with the matrix protein

                                                                         v.      L (RNA polymerase)

1.      Carries out all reactions necessary for capping and synthesis of viral mRNAs

2.      Bind the RNA genome in the virion

                                                                       vi.      Leader Sequence

1.      inhibits both RNA polymerase II and III

2.      enters the nucleus and may bind cellular proteins

d.      Single-cell reproductive cycle

                                                                          i.      Virion binds cell receptor (?) can enters the cell via receptor-mediated endocytosis

                                                                        ii.      The virion membrane fuses with the endosome membrane

                                                                      iii.      Viral nucleocapsid is released into the cytoplasm

                                                                       iv.      The (-) strand is copied into five subgenomic  mRNA strands by L and P proteins

                                                                         v.      N, P, L, and M proteins are synthesized on ribosomes in the cytomplasm

                                                                       vi.      G protein is synthesized by ribosomes attached to the ER, enters the secretory pathway and is transported to the plasma membrane

                                                                     vii.      N, P, and L proteins synthesize a full length (+) strand that serves as template for genomic RNA synthesis

                                                                   viii.      N and P-L associate with genomic RNA

                                                                       ix.      Matriz protein binds N and facilitates virion assembly by also binding protein G at the plasma membrane

                                                                         x.      The virion buds from the host cell basolaterally

e.      Disease

                                                                          i.      Symptoms

1.      Flu-like Illness in humans

2.      Acute infection producing vesicular lesions in cattle, pigs, and horses

                                                                        ii.      Transmission

                                                                      iii.      Mechanism

1.      CD4 T cells can accomplish non-cytolytic clearing of the virus

                                                                       iv.      Treatment Strategies

1.      Used as a viral vector in vaccine development to deliver high titers by a mucosal route

                                                                         v.      Detection

 

Togaviruses

A.     Genus

a.      Alphavirus (Sindbis Virus)

b.      Rubivirus (Rubella Virus)

B.     Example: Sindbis Virus

a.      Genome Organization

                                                                          i.      (+) strand RNA

                                                                        ii.      Genomic RNA has a 5’cap and 3’ poly-A tail

b.      Enveloped

c.      Function of Viral Proteins

                                                                          i.      Capsid Proteins

                                                                        ii.      E1 protein

1.      Envelope protein

2.      Forms a dimer with E2 in the ER

                                                                      iii.      E2 protein

1.      Envelope protein

2.      Forms a dimer with E1 in the ER

3.      Has a palmitate modification

                                                                       iv.      E3 protein

1.      Envelope protein

                                                                         v.      6K

1.      Envelope protein

                                                                       vi.      nsP1

1.      Part of (+) strand polymerase

                                                                     vii.      nsP2

1.      Part of (+) strand polymerase

2.      Protease

                                                                   viii.      nsP3

1.      Part of (+) strand polymerase

                                                                       ix.      nsP4

1.      Part of (+) strand polymerase

2.      Encodes the RNA-dependent RNA polymerase

                                                                         x.      P123 and nsP4

1.      (-) strand RNA polymerase

d.      Single-cell reproductive cycle

                                                                          i.      Virion binds to a high affininty laminin receptor on a host cell

                                                                        ii.      Virion enters cell via receptor mediated endocytosis

                                                                      iii.      Upon acidification of the endosome, viral RNA is released into the cytoplasm

                                                                       iv.      Viral (+) RNA is translated into the P1234 polyprotein and cleaved at various sites by nsP2 to form different RNA polymerases

                                                                         v.      P123 and nsP4 copies viral RNA into (-) strand RNA

                                                                       vi.      nsP1, nsP2, nsP3, and nsP4 copy viral RNA into (+) strand RNA and subgenomic mRNA

                                                                     vii.      Subgenomic mRNA is translated on free ribosome in the cytoplasm until the Capsid protein is released by proteolytic cleavage

                                                                   viii.      Cleavage of the capsid protein exposes a hydrophobic N-terminal domain of PE2 and the ribosome associates with the ER

                                                                       ix.      PE2, K6, and E1 proteins enter the secretory pathway and are brought to the plasma membrane

                                                                         x.      The capsid protein associates with viral (+) full length RNA and brings it to the plasma membrane by associating with viral glycoproteins

                                                                       xi.      The virion buds from the host cell

e.      Disease

                                                                          i.      Symptoms

1.      Subclinical

                                                                        ii.      Transmission

1.      Trasmitted by the bite of a Aedes mosquito

                                                                      iii.      Mechanism

1.      Bite by Aedes Mosquito

2.      Virus enters bloodstream (viremia)

3.      Antibodies in blood limit spread by viremia (IgM and IgG)

4.      Cell-mediated immunity is important to resolve infection

                                                                       iv.      Treatment Strategies

1.      No antiviral drugs

2.      Rubella (Rubivirus) has a live, attenuated vaccine

                                                                         v.      Diagnosis