CLASS: 9:00-10:00
DATE: 11-1-10
PROFESSOR: Dr. Lefkowitz
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XIV.
Negative Sense RNA Viruses
Scribe: Eric Larson
Proof: Lauren Morris
Page 1 of 7
Title [S1]
Contact Info [S2]
Objectives [S3]
a. The objectives of the lecture is to first go through some common and distinguishing characteristics of RNA
viruses in general
b. Discuss basic replication mechanisms of RNA viruses – how they produce some of their proteins and from
there cause disease
c. And finally discuss each family of viruses that make up this group, and talk a little bit about their pathogenesis
and disease in humans
Reading [S4]
References [S5]
Virus Classification [S6]
a. Ok so: virus classification
The Virosphere[S7]
a. This is a depiction of the world of viruses
b. The virus we’re interested in are the negative sense single strand viruses
The Virosphere [S8]
a. Here we see the group of single stranded RNA viruses
b. It’s worth pointing out, that in this group of viruses there are organisms that infect plants, invertebrates, and
vertebrates
c. Most of the organisms that infect vertebrates can also infect humans, and these are the viruses we’re
interested in
Features Used to Classify RNA Viruses [S9]
a. Here are the features we’re interested in for RNA viruses
b. We’re interested in viron symmetry: the virus can be icosahedral, and helical
c. The viruses may or may not have a lipid envelope derived from the host cell, and if they don’t, you’ll see them
as naked icosahedral capsids
d. The size of the particles are generally characteristic
e. The genome structure is especially characteristic for viruses
f. While the previous characteristics don’t necessarily indicate specific origins, genome structure is telling in
discerning the origins and properties of viruses
RNA Virus Genome Structure[S10]
a. Most RNA viruses are single stranded, some are double stranded
b. Strand polarity can be positive or negative
c. The segments are often single, but others have one, two, or up to nine segments
Single Strand Virus Genome Polarity [S11]
a. So when we talk about polarity, that is the characteristic which distinguishes the RNA viruses you studied last
week from those we are covering now
b. Plus sense RNA can be directly can be directly translated into a protein
c. Negative sense mRNA must be first transcribed into positive sense before it can be translated into a protein
d. All the viruses we discuss today are negative sense RNA viruses, though ambisense RNA genomes are a
little different
Negative/Ambisense RNA Viruses [S12]
a. Let’s now talk about particular groups of this class of viruses
Properties of Negative-sense RNA viruses [S13]
a. So let’s talk about properties
b. They all have an eveloped virion derived from the host cell
c. They all have helical nucleocapsids inside that envelope
d. Some are negative sense single segment RNA
e. Some are negative sense and ambisense multi segment RNA viruses
f. They generally all replicate in the cytoplasm, but there are some exceptions such as Bornaviruses, and
orthomyxoviruses which both have a component of the replication cycle that directly requires the nucleus
g. The genomes themselves are not infections – that is they must be first transcribed into positive sense RNA
before they can begin to produce new viruses
h. That’s different from the positive sense RNA viruses where you can introduce the RNA genome directly into
the cell and make viruses
Classification [S14]
a. So how do we classify some of these viruses?
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 2 of 7
b. Negative sense single strand viruses are part of the Mononegavirales order
c. That order is made up of several different families: bornaviruses, filoviruses, paramyxoviruses and
rhabdoviruses
d. All of these viruses here, if you compare their proteins, you can see that they all have a common ancestor
e. However, they happen to cause different types of diseases, despite their relation
XV.
Multi-Segment Negative and Ambisense ssRNA viruses [S15]
a. Ambisense viruses are a combination of both positive sense and negative sense RNA
b. They copy information off of both positive sense and negative sense segments
c. Arenaviruses and Bunyaviruses are two examples of ambisense viruses
d. They not natural pathogens of humans - you get them from bites of ticks or mosquitos
XVI.
Multi-Segment Negative Sense ssRNA Viruses[S16]
a. Finally, we’ll talk about orthomyxoviruses
b. They are all negative sense viruses, but these all have multiple segments that encode their proteins
XVII. Viral Replication Cycle [S17]
a. What are the features of a replication cycle that allows these viruses to produce progeny?
b. I’ll give a general overview of the while replication cycle
XVIII. Viral Replication Cycle [S18]
a. The virus must first attach, enter the cell, and uncoat their nucleic acids and any associated proteins into the
cell
b. At that point, if the virus is a positive sense RNA virus, translation can begin immediately
c. If the virus is a negative sense RNA virus you must first transcribe positive sense mRNA before translation
can begin
d. After translation, you now have enough proteins that you can start assembling into new virions
e. Assembly is inside the cell cytoplasm itself or at the membrane of the cell
f. In essence you have association with virion proteins with virion DNA
g. They then leave by budding off and picking up part of the cell membrane on the way out
h. Lot’s of detail, but this general scheme is about all you’ll need to know
XIX.
Diagram of the Viral Replication Cycle [S19]
XX.
ViralProteins [S20]
a. There are a number of different viral proteins
b. The different viruses mix and match combinations of these proteins depending on their functions
c. Important thing is: they all need proteins for attachment, proteins for the structure of the virus, and proteins
that mediate replication
d. For attachment, you might have the membrane glycoprotein, you might have a fusion protein and a
hemagglutinin, and you also may have a neuraminidase
e. All these viruses have a matrix protein which underlies the lipid bilayer and gives structure to the virion itself
f. Finally, there are a number of proteins required for replication, which we’ll discuss in more detail soon
XXI.
Rhabdovirus Virion [S21]
a. Rabies virus is a characteristic Rhabdovirus
b. For this virus, we can see how those proteins form up to give its characteristic structure
c. You have a lipid bilayer, you have G glycoprotein spikes coming out of it responsible for attachment
d. Underneath you have matrix responsible for the virion structure
e. Within that you have viral genome that has attached to it some of the replication proteins which I’ll talk about
in a second or two
XXII. Virus Coding Strategies [S22]
a. The simplest mechanism for coding is to have individual open reading frames (ORFs)
b. But these viruses have a lot of tricks to maximize the information that can be derived from their single, small
genome
c. So in addition to individual ORFs they might translate one large protein, which is then cleaved into two or
more individual proteins – that is polyprotein processing
d. Also, when they transcribe some of their DNA, they play with the sequence of the RNA itself by adding or
deleting bases to shift the reading frame one direction or the other which will produce a different protein
e. They also have multiple ribosomal initiation sites which means that by having the ribosome bind to different
sites, they can produce different proteins
f. Some have a stop codon read-through, which means that in some cases the codon causes a stop of the
translation, while in other cases it might be skipped over to give a different protein product
XXIII. Genome Organization Mononegavirales [S23]
a. This gives you an idea of what the genome organization looks like
b. This is for the order Mononegavirales
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 3 of 7
c. You can see how all these related viruses have similar proteins in their genomes
i. They all have the same proteins such as M and P proteins for replication, a matrix protein, a glycoprotein,
and they have a polymerase protein
You can also see here that all these proteins are in the same general order in the genome
ii. This is one piece of evidence that leads us to believe they have a common evolutionary origi
XXIV. Virus Replication Machinery [S24]
a. Here’s the replication machinery
b. You have RNA dependent RNA polymerase (RdRp) that reads the RNA and can either produce mRNAs for
proteins or virion RNAs
c. There’s a nucelocapsid protein which is what makes the nucelocapsid
d. That polymerase protein is dependent on the P protein for proper translation – it acts as a cofactor
e. The P protein binds to both the polymerase (L) and the nucleocapsidprotein (N)
XXV. RNA-dependent RNA Polymerase [S25]
a. The RdRp is the catalytic subunit of the polymerase complex that brings in nucleotides and allows for
elongation of the transcribed or replicated RNA genome
b. Transcription and genome replication are functions which are also associated with RNA polymerase, and it
can also have functions like capping, methylation and polyadenylation
XXVI. Source of the RNA-dependent RNA polymerase [S26]
a. Some viruses might need proteins contained in the host cell in their replication process
b. One protein no host cell has the code for is the RNA-dependent RNA polymerase
i. Host cells don’t code for this, so the virus is dependent on its own RNA dependent RNA polymerase to
surve
c. So the virus must provide that polymerase in one of two ways:
i. For positive strand RNA viruses, the virus only has to carry in the RNA genome which can be translated by
the host ribosome
ii. Since the negative strand can’t be directly translated, the virus must bring in the polymerase with them so
they can initiate mRNA transcription once it reaches the host cell
XXVII. VSV Transcription and Replication [S27]
a. Overview of VSV transcription and replication cycle of a prototypic virus
b. So at top you have host genome that codes for N P M G and L proteins
c. Transcription is initiated to produce mRNA
d. This begins with transcribing the code for N, and once it finishes transcribing there the polymerase terminates
and reinitiates at the start of the P code
e. So you don’t have the drop off of the polymerase, but just the termination and reinitiation of the same
polymerase at the junction of each subsequent reading frame
f. This will lead to the production of N proteins which encapsulate the virion RNA
g. Once you get enough N proteins produced you can begin replication
i. So initially we have transcription of mRNA – the switch to replication occurs when enough of the N protein
has been produced
h. In replication, you don’t have the termination-reintitiation process of transcription but rather you just make the
whole transcript continuously
i. So this is the process for VSV, but it works similarly for all the viruses we discuss today
XXVIII. Important Human Pathogens from the Group of Negative Strand RNA Viruses [S28]
a. Now we’ll talk about each individual group of viruses and their pathogenesis
XXIX. Major Target Tissues [S29]
a. In general, these are the major targets of viruses
b. Just want to point out some of the negative-sense RNA viruses
c. You have different types of viruses that infect the various target tissues
d. Moth of the viruses we’ll look at here are paromyxo and orthomyxo viruses
i. These are human pathogens in that they spread human to human without animal reservoirs
XXX. Oral and Respiratory Viral Diseases [S30]
a. These are the diseases some of these viruses cause
b. Notice a lot of respiratory diseases caused by paramyxo and orthomyxo viruses
XXXI. Arboviruses and Zoonoses [S31]
a. Arboviruses are viruses that have an insect vector that transmit them from their normal animal host to
humans
b. Zoonoses are viruses that humans are infected of as result of a contact of an animal – you pick it up directly
from animal rather than through the insect vector
XXXII. Arenaviruses/Bunyaviruses [S32]
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 4 of 7
a. Let’s talk about some individual viruses now
XXXIII. Arenavirus and Bunyavirus Disease [S33]
a. These cause disease by accidental contact with animals, or some are transmitted by contact with insect
vectors
b. The diseases are often seen in Africa – rare in U.S. and Europe, but people traveling can be infected by
these
c. Among the arenaviruses is the junin virus which causes hemorrhagic fever
d. Again, with the bunyaviruses the main disease is hemorrhagic fever
i. One type is the Hantaviruse (comes from contact with rodent droppings) which does occur in the U.S.
e. You can get serious hemorrhagic fevers from all these viruses
XXXIV. Arenavirus Genome Organiation [S34]
a. So for ambisense viruses, some parts of virion mRNA must be transcribed before it can be translated, and
even though other parts of the RNA appear as if they can be directly translated (like + sense), they must be
first transcribed before they can be translated as well
b. Arenaviruses have two segments: large and small segments
c. On their genome they have various proteins which we’ve seen before
d. They have polymerase protein (L), and N protein which encapsulated the virion RNA
e. Glycoprotein used to attach to the cell
XXXV. Bunyavirus Genome Organization [S35]
a. Bunayavirus again has multiple segments – a small segment, a medium segment, and a large segment
b. Various segments acquired by cleaving of polyprotein
c. All of these segments are negative sense, although some of them look a little similar to the ambisense
arenavirus RNA
XXXVI. Filoviruses [S36]
a. So there are very few of Bunyaviruses and Arenoviruses that are purely human viruses (few that don’t have
an animal host or insect vector)
b. Filoviruses is another group of viruses that aren’t normal human pathogens
c. They have a characteristic, long, extended virion structure
XXXVII. Filovirus Disease [S37]
a. The mortality of these viruses is close to 100%
b. Very deadly – cause severe hemorrhagic disease in which bleeding is characteristic
c. The two main groups are Marburg virus and Ebola virus
d. Bats are thought to be the primary animal reservoir of these viruses, although humans or other animals might
become infected through contact with bats
e. Once a human is infected, you get an outbreak and the spread is human to human, but usually that outbreak
dies out after a few weeks or months
XXXVIII.
Filovirus Virion and Genome Organization [S38]
a. Here is the genome organization of these filoviruses
b. Filoviruses is one of those viruses that uses multiple mechanisms for getting extra info out of its genome
c. Filoviruses use polyprotein to get multiple proteins from one long protein
d. They also use RNA editing - while you’re transcribing the G protein there might be residues added or
removed from the coding sequence which forms different proteins
e. Those resulting proteins have numerous roles in the virus, some of them we know, others are still unknown to
us
XXXIX. Filovirus Pathogenesis [S39]
a. They cause severe homorganic disease and bleeding because they target the blood vessels and that’s then
when you get the vascular injury and such
XL.
Rhabdoviruses [S40]
a. Ok, so now we look at Rhabdoviruses
b. One we know well is the rabies virus
XLI.
Rhabodoviruses [41]
a. VSV is a common laboratory strain of virus that is used to study this whole group of viruses
b. Rabies virus is the virus we’re most concerned with
c. Again, humans are the accidental host after coming in contact with infected animals
XLII.
Untitled [42]
a. Rabies virus causes characteristic neurological symptoms; foaming at the mouth and such
b. Usually you’re infected by contact (often bite), which allows the virus to travel through the nervous system to
the brain
c. Once it reaches the brain, you get the characteristic neurological symptoms
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 5 of 7
d. Rabies, also, is another one of these viruses with a high mortality
e. It’s thought that inducing a coma might be an effective way to treat rabies virus
i. This would reduce brain activity and also slowing down virion replication, but there is debate as to the
effectiveness of this method
XLIII. Paramyxoviruses [S43]
a. Most of the viruses we’ve looked at up til now have not had humans as the primary host, but the
paramyxovirus has a large number of human pathogens
b. They can infect animals as well, though
XLIV. Paramyxoviruses [S44]
a. See how it can infect humans, but also animals
b. Often the virus stays with the organism it originally develops in – that is, there’s not much cross-infection
between animals and humans
XLV. Paramyxovirus Genome Organization [S45]
a. Have the characteristic NM attachment protein, the polymerase protein, and other proteins common in this
group of viruses
b. Paramyxovirus also like to use tricks to maximize their proteins
i. See how one ORF reads “P/C/V” – that’s because you can get P protein, C protein, or V protein from that
one ORF
XLVI. Paramyxoviruses: Maximizing Genetic Content [S46]
a. What they do here is a number of things
b. Some of the viruses use mRNA editing – take an ORF and edit the mRNA to get different protein products
c. Some use ribosome choice – in one reading frame there might be various parts the ribosome can bind, which
also affects the products produced
XLVII. Human Respiratory Syncytial Virus [S47]
a. This is a virus that has infected almost all of us as kids
b. In general cases it is a mild cases
XLVIII. Measles Virus [S48]
a. Measles virus is a concern for humans
b. f you aren’t vaccinated against it, you can get the characteristic rash, a subsequent acute infection and then
recover from it
XLIX. Neurologic Complications of Measles [S49]
a. However, then there are types of encephalomyelitis that can occur after the initial measles infection which
can be very serious
L.
Orthomyxoviruses [S50]
a. Now we’ll look at orthomyxoviruses and how they cause disease
LI. Orthomyxoviruses [S51]
a. We’re mostly concerned with influenze A and B viruses
b. There are other viruses, but those don’t come up often
LII. Schematic Diagram of Influenza A Viruses [S52]
a. They contain mutli-segment genomes
b. Each genome encodes one, or maybe two proteins
LIII.
Influenza A Genome Structure [S53]
a. When we look at the genome, we see they have the typical set of proteins (L protein, matrix protein,
attachment proteins)
LIV.
M2 Ion Channel [S54]
a. Some of the important proteins:
b. M2 ion channel protein is one of the unique proteins
c. That ion channel can be target and inhibited to help prevent influenza infection
LV.
Hemagglutinin [S55]
a. Hemagglutinin is a protein that functions to bind the virus to the cell, and that is often targeted by antibodies
which can then prevent infection
LVI.
Neuraminidase [S56]
a. So the hemagglutinin binds to the host cell, but in order for the virus to be released from that cell you need a
protein to break that bond
b. Neuraminidase is the protein which breaks that bond
c. Target of various drug treatments
LVII.
Influenza Virus – Variation and Evolution [S57]
a. So we all hear about variations of different viruses
b. These viruses have altered amino acids, which are recognized differently by antibodies
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 6 of 7
c. If one virus has enough changes from a similar virus you have an existing immunity towards, it won’t be
recognized by your antibody
LVIII. Influenza Virus Variation [S58]
a. Influenza virus has a number of mechanisms that allow it to change its amino acids so that it can avoid
immune response
b. You get an antigenic shift which is a mixing of components of genome segments from a different virus strain
c. You can also get recombination in which the same segment recombines with another segment of another
virus, but this is much more rare in influenza viruses
LIX.
Reassortment [S59]
a. Looking more closely at antigenic shift
b. Two viruses infect the same host, both of their genomes are present in the host, so when you get new virions
produced you can get new combinations of genome segments arising in the produced viruses
LX.
Emergence of Pandemic Influenza Viruses [S60]
a. This shows a brief overview of influenza virus in humans, starting back at 1918 with the outbreak of a
particular H1N1 virus
b. Over the years, this initial H1N1 virus has changed and picked up components of other viruses
c. You continue to get new segments, mixing and matching them constantly, which is what leads to the
constantly changing types of viruses we see from year to year
LXI.
US Influenza Surveillance 2004-2008 [S61]
a. See these characteristic peaks of viruses throughout the year which rise and fall based on the numbers of
people infected throughout the year
b. You get different proportions of the different viruses, and you get different strains of the various viruses which
have varying susceptibility to vaccines we’ve developed
LXII.
US Influenza Surveillance 2008-2009 [S62]
a. The spring outbreak of this past 2008-2009 was extremely unusual – this was the popular H1N1 epidemic we
recently observed
LXIII. Genesis of Swine-Origin H1N1 Influenza Viruses [S63]
a. That virus (from previous slide) was found to have segments of genome from four different sources – swine,
human, avian, and second swine virus were all contributing to the virus which resulted in a worldwide
epidemic
b. It had two different swine virus components, avian virus components, and a human virus component which
resulted in a complex virus which caused that worldwide epidemic we saw recently
LXIV. Why Pigs? [S64]
a. Influenza viruses are tuned to the specific receptors of the species of host they infect
b. Pigs are susceptible to infection by both avian and human viruses, so they act like mixing bowls for different
strains producing all sorts of new viruses which can reinfect humans and birds
LXV. US Influenza Surveillance [S65]
a. The normal seasonal flu here almost entirely consisted of the H1N1 flue
LXVI. Flu Surveillance October, 2010 [S66]
a. This is the flu outbreak just beginning this year, so we don’t have much information yet
LXVII. Fighting Back [S67]
a. Luckily, we do have vaccines and antiviral drugs like tamavir and rolenza against neuramindase and
amantidines which are ion channel blockers
LXVIII. Antiviral Resistance, 2008-2009 Season [S68]
a. Antiviral resistance in 2008-2009 showed different primary strains that had different drug resistances
b. Resistances change from year to year, and so you have to see which drugs are effective every season
LXIX. Vaccine Development [S69]
a. Vaccines are consistently the most effective treatment against viruses
b. Their components change from year to year by looking at what was infecting people towards the end of the
previous flu season and using that to compose a vaccine for the upcoming flu season
LXX. Vaccine Strains for the 2008-2009 Season [S70]
a. That H1N1 pandemic virus was not present in the vaccine for the 2008-2009 season, because it was not
around the year before
LXXI. Vaccine Strains for the 2008-2009 Season and the 2010-2011 Seasons [S71]
a. This year’s vaccine does have H1N1 in it
LXXII. The reason [S72]
LXXIII. Untitled [S73]
a. I mentioned that pigs are mixing bowls for new influenza viruses that infect humans
CLASS: 9:00-10:00
Scribe: Eric Larson
DATE: 11-1-10
Proof: Lauren Morris
PROFESSOR: Dr. Lefkowitz
Negative Sense RNA Viruses
Page 7 of 7
b. The question is, how do humans get the viruses from the pigs? The picture on this slide is one possible
mechanism for that
c. Question: What does neuraminidase do?
d. Answer: The virus must attach to the cell in order to infect it, that attachment is by the hemagglutanin. That
attachment is pretty strong, so in order for the virus to release from the host cell the attachment must be
broken which is what neuraminidase does.
[End 51:12 mins]