an agent that is not itself alive (i.e. no metabolic machinery), but can reproduce itself within a living cell
a virus can exist as two different forms:
2. replicative form
form of a virus found outside a cell that contains genomic material surrounded by a protective protein layer called a capsid
it may also sometimes have an envelope, an additional outer lipid layer derived from cell membrane
its purpose is to carry genetic information of the virus from one host cell and one host organism to another & to protect it from the depredations of the external environment
replicative form of virus
exists in such a form when it is inside the cell
its purpose is to direct the cell to make more virions and to ensure that the replicated virus is further transmitted to other cells and other hosts
eukaryotic mRNAs are monocistronic
as a rule, only one protein can be translated from each mRNA
ssRNA viral genomes
plus/sense: “normal” mRNA, usually with a polyA tail and can itself be used as a transcript for protein production
minus/antisense: complement to the sense strand, lacking a polyA tail & must be replicated in order for proteins to be made from it
replication of RNA requires enzymes not normally found inside host cells
How does the viral genome migrate to the proper cellular compartment?
it brings a genome to the nucleus for replication and transcription or causes the genome to associate with an intracellular membrane
Why study viruses?
1. they're important agents of human disease
2. they're important models for cellular functions (esp. gene expression)
3. they're important models for human disease, even if not caused by human viruses (animal models of virus induced cancers led to oncogene discovery)
4. provide new tools for biotechnology & gene therapy
the viral DNA is translated to make a single large polyprotein; the genome has one single coding region that results in a number of different proteins stuck together when initially translated
one protein in the polyprotein, usually a viral protease, can cleave different proteins out of the conglomerate
eg. poliovirus (picorna)
How do viruses overcome the inability of eukaryotic cells to translate more than one polypeptide from a single mRNA?
1. differential splicing of a pre-mRNA
2. synthesis of a polyprotein followed by autoproteolysis to release individual polypeptides
3. carriage of a segmented genome
segmented viral genome
the viral genome is broken up into multiple parts which code for different mRNAs which can be translated into separate proteins
each segment of genome/the subsequent mRNA gives rise to distinct protein via translation
and together they make up the whole genome
eg. influenza (myxovirus)
under viral control a single genome is preferentially transcribed to give separate mRNAs that code for individual proteins (the mRNAs come from a single genome though)
differential splicing viral genome
a phenomenon particularly present in DNA viruses, where it uses the host splicing machinery to differentiate mRNAs from a single DNA strand
in vitro Translation Assay
the purified genome (genetic material ONLY) is added to a tube with ribosomes, tRNA, & buffer
PROTEIN can only be made by viruses who are + stranded RNA so they can be used as a template in their original form
Enzymatic Activity Assay
the virion is added to ETHER, CHCl3, or a mild detergent that gently bursts the virion, so both it's genetic & proteinaceous contents remain intact
only thing added are dNTPs, NTPs, & buffer
NUCLEIC ACID TRANSCRIPTS (dna or rna) are only made if the virus carries ITS OWN transcription enzymes
1. myxovirus: will make RNA *IF there is mRNA in the assay so it can steal their 5' caps
2. retrovirus: will make DNA
determines whether a purified genome is infectious
the virus must be able to use transcription materials (enzymes) from a host cell, and not need their own
1. papilloma virus
genomes are purified from a virus & introduced into cells
with viruses that do not require virion enzymes (DNA & + RNA) a complete infection cycle can be started in this way
small viruses with an + strand (mRNA-like) RNA genome
they do not carry their own enzymes, but use host transcription enzymes
the capsid structure = icosahedral & they are NOT enveloped (nekkid)
they lyse cells
they make protein in an in vitro assay & are infectious (replicate) in a transfection assay but cannot replicate in an enzymatic activity assay
eg. Poliovirus, Hepatitis A, Foot-&-Mouth, Rhinovirus (common cold), Cardiovirus, Coxsackie, + other “enteroviruses” cause gastrointestinal discomfort + “stomach flu” symptoms
are transmitted fecal-orally
Picornaviruses' + strand RNA have a polyA tail at the 3’ end and what at their 5' end?
a small terminal protein called VpG (Viral Protein G) covalently bound to the 5' end
besides capping the 5' end of the transcript it also serves as a primer for host DNA pol to replicate the virus
What does the picornaviruses + strand RNA genome code for?
it contains one large open reading frame which gives rise to one polyprotein that is subsequently cleaved to make all the necessary viral proteins
this virus never enters the nucleus - stays in the host cell cytoplasm and can be translated by ribosomes there
the virus infects cells via interaction with CD155, a SPECIFIC cell surface protein receptor
CD155 is normally used to establish intercellular adherens junctions between
What limits the ability of the Picornavirus to infect some kinds of cells?
the distribution of the receptor CD155, as well as the fact that only some species of animals (i.e. primates) have it
How does the Picornavirus enter cells once bound to CD155?
it is endocytosed
the capsid fuses with the cell membrane, releasing the genome into the cell’s cytoplasm
there the genome associates with cellular polyribosomes, is translated, & gives rise to the viral polyprotein
What part of the cell is not necessary for a picorna virus infection?
the cell's nucleus is not necessary for picornavirus replication
because the genome is + strand RNA there is no need for it to travel to the nucleus
studies have been done where the cell nucleus is removed and polio infection is still successful
What happens to the viral polyprotein once it exists?
it's cleaved into 4 general sections
1. replicase: copyies more viral genome
2. accessory proteins: such as VpG
3. protease: cleaves the polyprotein
4. VPs (virion proteins): assembled as a precursor for an empty capsid, which is later made into a virion; there are 4 virion proteins (VP 1 -> 4) that are only cleaved from one another after assembly of the capsid
What is the structure of the picornavirus capsid?
the capsid is an arrangement of 60 protomers in a tightly packed Icosahedral structure
each protomer consists of 4 polypeptides known as VP (viral protein)1, 2, 3 & 4
VpG is the primer for replication so 1st it associates w/ the 3’ end of the + strand RNA template
replicase copies the entire plus strand, leading to production of a full length – strand RNA template w/ a poly(U) sequence at its 5’ end*
VpG then associates with the 3’ end of the – strand, & the same process is used to make another + strand
What is absent from the – strand picornavirus RNA genome once it's been copied from an origininal + strand RNA template by replicase?
the minus strand lacks a polyA tail
instead has a complementary poly(U) sequence at its 5’ end
all of the RNA (- & +) have VpG at their 5’ end, but only + strand RNA have polyA tails
+ strands: be used as mRNAs, templates for further replication, & as genomes
– minus strands: ONLY be used as templates for replication; are more efficient templates though, are better at directing synthesis of + strand RNA
picornavirus assembly & release
when enough uncleaved virion proteins (VP 1 -> 4) are made they're assembled into capsids
capsids are stuffed w/ + strand RNA & the precursor
proteins are cleaved -> a very tight strong structure is cemented
the virion is released by lysis of the host cell (picornaviruses are naked, so budding is NOT the method of release)
Which type of viruses are the only group to lyse host cells?
virus whose genome is minus (-) strand RNA
the only test in which myxoviruses will show a positive result is an enzymatic activity assay *as long as mRNA are thrown into the mixture so the virus can steal their caps
carry transcriptase in their helical nucleocapsid
they are have an envelope
eg. influenza, fowl plague
What is the only virus that has a helical nucleocapsid structure?
all others (papilloma, picorna, & retro) have a icosahedral shaped capsid
a very large group of viruses that all cause influenza-like disease
it's not naturally a human virus; found predominantly in wild waterfowl populations & occasionally makes its way into domestic animals or humans
eg. 1918 influenza epidemic (worst pandemic in the history of the world # of deaths-wise) or the recent H1N1 pandemic
myxo in Greek = “slime”; myxoviruses are often found in slimy environments
an enveloped viruses containing single, - strand RNA
on the envelope are 2 glycoproteins, HA & NA
within the envelope are 8 helical capsids, or nucleocapsids, that each contain a – RNA genome segment, transcriptase, & nucleoprotein (NP) protein
a myxovirus genome is segmented amongst the 8 nucleocapsids
the virion contains also contains Matrix Protein
protein that binds sialic acid (N-acetyl neuraminic acid), a carbohydrate often found as the terminal sugar on glycoproteins
sialic acid is the receptor for myxovirus (how they enter cells)
HA can cause hemagglutination, aggregation of RBCs b/c RBCs contain sialic acid on their cell surfaces
as assay that can detect a myxovirus
mix virus with a preparation of RBCs; these have sialic acid on their surface
if myxovirions they will interact with RBC surface and give rise to a clump/precipitate of RBCs = agglutination reaction
agglutination reaction can be blocked by adding an antibody for the virus --> Ig binds virus, making it unable to bind RBCs' sialic acid, won't see clump
can be used as asimple way of testing peoples' serum to see if they've been exposed to the same virus
an enzyme that destroys the sialic acid cell receptor so that newly made virions can break free from agglutination
also prevents other viruses from entering the host cell, as myxoviruses work a little slowly
What is within the 8 nucleocapsids of a myxovirus?
1. a segment of the – strand RNA genome (the entire genome is segmented amongst the 8 helical capsids)
3. Nucleoprotein (NP)
an enzyme that catalyzes the synthesis of a complementary RNA molecule using an RNA template
doesn't exist in eukaryotic cells
a component of nucleocapsids that always stays attached to a viral genome/template
it initiates the switch from transcribing mRNA from the - strand RNA genome to making + strand RNA from which more - strand RNA genome can be made for packaging and viral release
Matrix Protein (M1)
links the outer envelope to the inner nucleocapsids
the virus infects cells via HA interacting with the terminal sialic acid found on the end of many cell surface proteins
it acts as a receptor, and once bound, the virus is endocytosed after a drop in pH
once the virus is inside the 8 helical nucleocapsids move to the nucleus
What initiates the fusion of the myxoviral envelope and the cell membrane?
a drop in pH
How and where is myxoviral mRNA synthesized?
viral transcriptase reads the – strand RNA genome as a template inside the nucleus to produce complementary viral mRNA
5' caps are stolen from newly made host cellular mRNA as primers
the PolyA tails are added using a “stuttering” mechanism
the newly synthesized viral mRNA is transported back to the cytoplasm where translation of the necessary viral proteins begins
Which viral proteins are synthesized on MEMBRANE-bound polyribosomes and which are synthesized on FREE polyribosomes?
membrane-bound: HA + NA
free: M1, NP, transcriptase, & other internal proteins
HA, NA, & M1 are sent to the cellular membrane, while all other internal VPs are sent to the nucleus to aid in replication
What does accumulation of enough synthesized nucleoprotein do?
it signals a switch from making mRNA (w/ stolen caps & a poly-A tail) to making EXACT plus strand copies
*Nucleoprotein (NP protein) initiates & signals for this switch to occur
these are then used as templates to make MORE minus strand genome
mRNA --> + strand RNA --> - strand RNA genome
What happens when the myxovirus is ready to assemble itself and be released from the host cell?
NP protein & transcriptase have been associated w/ the genome during replication all along in the nucleus
complete nucleocapsids (genome, NP, transcriptase) are transported to cytoplasm
in cytoplasm they associate with M1 protein which itself is partnered w/ cytoplasmic ends of HA and NA inside the cell membrane
*the virus is released by budding, resulting in an ENVELOPED virus & a perfectly intact host cell
What made HeLa cells so aggressively malignant?
that they contained – and continuously expressed – several copies of a partial DNA molecule from human papillomavirus (HPV) type 18, the cause of the cancer and death of Henrietta Lack some 30 years previously
a benign, protruding, epithelial tumor (eg. wart)
papilla = a nipple-like protrusion
associated with warts on many parts of the body
are many types of papillomaviruses
almost all cause only common, benign, self-limiting infections of epidermal tissue
a few (eg. HPV 16 & 18) are the major causes of cervical, penile, & throat cancer
vaccines are now available & are quite effective at preventing infection and cancer if administered prior to encounter
are naked, double-stranded DNA viruses with an icosahedral capsid made up mostly of L1 protein in association with a few molecules of L2 (coded for by genes of the same name respectively)
its genome is 8 kb of circular double stranded DNA (~picornavirus genome)
it contains early (E1-7) & late (L1-2) genes
What are the ONLY types of cells HPV can infect?
basal cells of the skin & mucosal tissue (epithelia)
its replication is tightly coupled to the development of these cells as they divide, move, differentiate, & eventually slough off
unlike picorna or mixo, HPV replication is very restricted
What 'receptor' does HPV use to get into host cells in the basal layer of the epidermis?
1. heparan sulfate proteoglycan: a nonspecific component of the cell membrane & extracellular matrix)
2. another, more specific receptor, that is unknown
By what mechanism does the papillomavirus enter the cell and which protein is required for the entry process?
after binding to the proteoglycan heparan sulfate, HPV enters the cell via ENDOCYTOSIS & once inside the cell passes through the endosomal membrane
entry requires participation of the L2 PROTEIN
once inside the cell the dsDNA virus travels to the nucleus where it uses HOST cellular machinery to aid in transcription & DNA replication
What is the goal of early HPV gene expression?
to make the gene products (viral proteins) necessary for genome replication
viral genome is originally transcribed by cellular RNA polymerase from the early promoter
the transcript is differentially spliced & translated to make a number of early gene products: E1 --> E7
forms a complex at the origin of DNA replication that, along with E2, recruits the host cell’s DNA polymerase machinery
E1 also serves as a HEL1CASE that unwinds the DNA, making it accessible to the replication complex
the master regulator of both transcription & DNA replication
it interacts w/ both promoters, the origin of replication, & the cell’s RNA synthesis machinery
E3 & E4
interact with various cellular components (including the cytoskeleton) to modify the cell structure and make it amenable to virus replication & release
E5, E6 & E7
critical viral proteins for altering the growth & DNA replication patterns of the infected cell
stabilizes (therefore activates) the receptor for epidermal growth factor
this constitutively stimulates the cell to divide
interacts w/ & causes the degradation of p53, preventing the infected cell from recognizing the viral DNA as foreign and thereby preventing apoptosis
[E6 prevents the cell from apoptosing itself]
think p5 … E6 … 3, 5 --> 6
binds to Rb, a central regulator of cell division, stimulating entry into S-phase DNA synthesis
excessive division of the infected cell not only leads to warts, but also helps to propagate the virus & to cause the cell to maintain high levels of DNA replication components (both enzymes & nucleotides) needed for viral genome synthesis
HPV DNA Replication
the double-stranded DNA genome is replicated using the normal cellular machinery, directed to the HPV replication origin by E1 & E2
HPV genome replication is coupled to division of the infected cell (unlike other viruses whose genomes replicate exponentially)
As basal epithelial cells divide, differentiate, & migrate toward the epidermal surface, why does the pace of viral DNA synthesis greatly increase?
so that large numbers of virions can be present in terminally differentiated keratinocytes that make up the outermost layer --> when they die naturally lots of virions will be released
up until the final stages of terminal differentiation the amount of viral DNA is maintained at about 100 copies per cell
What is the goal of late gene expression in the papillomavirus?
to make the L1 and L2 proteins necessary for virion assembly
similar to early gene expression, late gene expression also uses differential splicing to make mRNAs for both proteins from a SINGLE primary transcript
At what cellular stage are HPV virions assembled and how are they released?
virions are assembled in the infected cells in the granular layer immediately below the outermost epithelium
they are released when dead, cornified cells containing lots of virions are sloughed off
(the virus can survive for a long time on surfaces, ready to be picked up by & infect the next person)
How can papillomaviruses be oncogenic/cause malignancy?
by a rare events during which a portion of the viral DNA including E1 & E6, but NOT E2, integrates into the host cell genome
in these cells, continued expression of the early genes will enable the cell to divide continuously out of control & become a cancer cell (the genetic change becomes heritable)
HPV, particularly high risk types (16, 18, + a few others) is a major cause of naturally occurring human cancer, particularly at genital sites
What happens if E2 doesn't integrate along with the rest of the viral genome? What happens if it does integrate into the host cell chromosome?
if part of the viral genome integrates without E2 (but say with E6 & E7 in a way they're expressed) there's NO control over the early genes promoter --> the cell replicates uncontrollably --> cancer
if the WHOLE genome integrates, E2 is present & still regulates the virus' expression --> wart but not cancer
[E2 is the MASTER regulator of transcription]
viruses whose information flows in the reverse of the usual pattern: RNA --> DNA (eg. HIV)
are enveloped, + strand RNA viruses w/ an icosahedral capsid
the envelope is derived from the host cell membrane modified by Env, a viral-encoded protein
contain MA, a matrix protein that connects the outer envelope to the inner capsid
Env is structurally similar to what other viral protein?
HA protein of myxoviruses (influenza)
What does a retroviral capsid contain?
TWO copies of the + strand RNA genome
the enzymes reverse transcriptase (RT), integrase (IN), and protease (PR)
the nucleocapsid (NC) protein
its capped, poly-adenylated, and has a tRNA base-paired near the 5’ end
tRNA acts as the primer for reverse transcription
the genome contains a minimum of 4 genes (gag, pro, pol, env) & has short, repeated sequences (R) at both ends
it has a unique sequence at the 5’ end (U5) & a different unique sequence at the 3’ end (U3)
How do retroviruses get into host cells?
they interact with host cells via the Env protein (found modifying the viral envelope) & a specific cellular receptor (CD4 on CD4+ T cells w/ HIV)
the virus enters the cell either by direct membrane fusion or receptor-mediated endocytosis, depending on the type of virus
Where does reverse transcription take place once the retrovirus has entered the host cell via membrane fusion or RME?
the genome remains in the cytoplasm for reverse transcription
the process initiates downstream of the 5’ end of the genome where the tRNA primer is located
RT moves TOWARD the 5' cap (to the left) and once it hits the end, the small piece of DNA made “JUMPS” to the 3’ end of the RNA, its RU5 sequence annealing to the U3R sequence
reverse transcription now proceeds toward the 5’ end again, completing the strand of “minus” DNA (complementary to the viral RNA genome)
a plus strand of DNA is made using the minus strand as a template --> dsDNA made
What is the structure of the final double-stranded retroviral DNA that is produced by reverse transcriptase in the cytoplasm?
the U3RU5 sequence = LTR (long terminal repeat)
the completed dsDNA is transported to the nucleus as part of the preintegration complex
How does the viral dsDNA integrate into the cellular DNA?
using the viral integrase enzyme
integration is irreversible
the genome is integrated at random sites, forming a provirus
the provirus is replicated w/ cellular DNA & passed on to daughter cells via mitosis
What transcribed the provirus?
CELLULAR RNA polymerase II, directed by a promoter located within the 5’ U3 region
transcripts are poly-adenylated after the 3’ LTR
if the mRNA is spliced, Env protein is translated on membrane-bound polyribosomes & sent to the cell surface
if the mRNA is NOT spliced, internal viral proteins (Gag, Pro, Pol) are translated by free ribosomes in the cytosol as polyproteins (Gag, Gag-Pro, Gag-Pro-Pol)
Retroviral Assembly & Release
polyproteins associate w/ the viral genome, then move to the inner cell membrane
as the capsids assemble, Env proteins incorporate on the outside of the membrane, & an immature capsid buds from the membrane
ONCE BUDDED, viral protease cleaves the viral polyproteins to produce individual Gag, Pro, Pol, changing the virion from an immature, noninfectious agent to a mature and infections virus
What do the individual Gag, Pro, and Pol proteins do?
they provide the virion w/ all the necessary replication enzymes (reverse transcriptase, integrase, & protease)
What do all protooncogenes encode even thought they're all very different from each other?
they all encode proteins critical for regulation of cell division
can be activated by:
1. retroviruses integrating into the genome adjacent to a protooncogene, causing it to be expressed under control of the viral LTR
2. protooncogenes becomming part of the viral genome; such viruses can transform all the cells they infect & cause cancer rapidly
3. means other than virus infection – chromosomal rearrangement & mutation
What are some targets of AIDS prevention or treatment regimens, as well as just general antiretroviral treatment?
1. inhibitors of viral DNA synthesis, re: reverse transcriptase
2. integrase inhibitors
3. Protease inhibitors
retroviruses whose genes (not counting oncogenes) code for virion proteins & their interaction with cellular functions (like transcription and splicing) is entirely PASSIVE
simple retroviruses can only infect, replicate, & cause disease in newborn or immunosuppressed individuals
retroviruses that encode additional proteins that interact w/ cellular proteins & participate directly in regulation of viral expression and inhibit functions critical to virus replication
a complex retroviral protein that activates transcription by interacting with TAR, a region near the 5’ end of the nascent HIV RNA transcript
this interaction leads to alteration of RNA polymerase by phosphorylation allowing continuation of transcription to the end of the provirus
a complex retroviral protein that regulates expression of genes for virion proteins by mediating export from the nucleus of incompletely spliced (gag-pro-pol & env) mRNAs
without Rev, only smaller, completely spliced mRNAs encoding Tat, Rev, & several accessory proteins are translated
Vif, Vpu, Vpr, Nef
bind to & neutralizing host cell proteins that can block virus replication at various stages of replication
they are accessory proteins that aren't essential for replication of HIV in cell culture, but play important roles in enhancing the efficacy of the virus
What is the overall function of Tat, Rev, & the accessory proteins?
they allow HIV to evade host adaptive and innate immune responses & replicate efficiently in immunoCOMPETENT adults
What is the only type of virus that can enter the cell via something other than endocytosis?
the retrovirus: it can enter by direct membrane fusion OR receptor mediated endocytosis
eg. HIV enters by direct membrane fusion
Which two types of viruses are enveloped and therefore do not lyse cells?
both contain a contain a matrix protein that connects the outer envelope to the inner capsid
Which kinds of cells are most often affected by which viruses?