Download General Replication Strategies for RNA Viruses

General Replication Strategies for RNA Viruses
Guest Writer: Dr. Michael Beard
Introduction
The replication of viral RNA genomes is unique considering that the host cell does not
contain a RNA dependent RNA polymerase. To overcome this constraint, the majority
of RNA viruses encode their own RNA polymerase that is either packaged with the
virus genome or is synthesised shortly after infection.
Single strand RNA viruses can be classified into three classical groups according to
the polarity of their genomes, which in turn dictates the strategy for viral replication
(White and Fenner, 1994). The first group, viruses with (+) sense RNA genomes
include the Picornaviridae, Flaviviridae, Togaviridae, Caliciviridae and the
Coronaviridae. The linear RNA genomes of the Picornaviridae and Flaviviridae are
polycistronic and act as mRNA that is translated into a viral polyprotein which is
subsequently cleaved into individual viral polypeptides, one of which is a RNA
dependent RNA polymerase. This polymerase uses the (+) sense input RNA as a
template for the transcription of (-) sense RNA which in turn can act as a template
for the production of nascent (+) sense RNA. This newly transcribed (+) sense RNA
has 3 possible functions;
(i) mRNA
(ii) a template for production of additional (-) strands or
(iii) packaged as progeny virus.
In contrast to the above replication strategies the Togaviridae and the Coronaviridae
use a slightly different mechanism in which subgenomic mRNA species are used for
the transcription of the structural proteins. Only the 5' two thirds of the Togaviridae
(+) sense genome is translated that results in the synthesis of a polyprotein that is
post- translationally cleaved into the non-structural proteins one of which is a RNA
dependent RNA polymerase. This polymerase then synthesises full length (-) sense
RNA from which two species of (+) sense RNA are copied;
(i) virion RNA that can be packaged or can act as a template for more (-) strand
synthesis
(ii) a smaller subgenomic mRNA species that is translated into a polyprotein from
which the structural proteins are derived.
The Coronaviridae replicate using a similar mechanism in which a subgenomic 3'
nested set of overlapping (+) sense mRNA molecules are synthesised for the
translation of the structural proteins. All the (+) sense RNA viruses share a common
theme in that their genomes all have the ability to act as a mRNA thereby
eliminating the need for the virus to package a RNA polymerase.
The second group, comprising the Orthomyxoviruses, Paramyxoviruses,
Bunyaviruses, Arenaviruses and Rhabdoviruses all have (-) sense RNA genomes. The
genomes of these viruses must serve two functions, firstly to serve as a template for
transcription to generate (+) sense RNA and secondly as a template for replication.
Upon entry into the host cell the (-) sense genome is transcribed to generate (+)
sense monocistronic RNA that serves as mRNA for the production of viral proteins
which initiate genome replication. This (+) sense RNA also serves as a template for
the synthesis of (-) strand genomic RNA's. In contrast to the (+) sense RNA viruses
described above, (-) strand viruses must package a functional RNA polymerase to
initiate transcription of their (-) sense genome.
The Retroviridae make up the third group in which the RNA genome does not
function as a (+) or (-) sense molecule but acts as a template for the production of
viral DNA. This is achieved by RNA dependent DNA polymerase (reverse
transcriptase) that is packaged with the RNA genome. The resulting viral DNA
integrates into the host cell genome to provide the template for viral RNA synthesis
by host derived mechanisms.
This article has been reproduced with the kind permission of its author, Dr. Michael
Beard, previously of the Department of Medicine, Divison of Infectious Diseases,
University of North Carolina.
This work may not be reprinted without the prior knowledge and consent of its
author.
In addition to the chemical differences we have already talked about here, RNA is also
shorter, and generally single-stranded (this does not apply to some viruses as we will see
later). RNA also functions differently from DNA.
While the only role of DNA is the storage of genetic information, RNA has many different
roles to fulfil. There are several types of RNA which perform these different functions.
Ribosomal RNA (rRNA) form complexes with protein to form ribosomes, the site of
protein synthesis wihtin the cytoplasm of the cell.
Messenger RNA (mRNA) carries the information recorded in DNA from the nucleus to
the cytoplasm of the cell.
Small nuclear RNA (snRNA) is involved in pre-mRNA splicing.
Heterogenous nuclear RNA (hnRNA) is the primary transcript from the eukaryotic
enzyme, RNA polymerase II. hnRNA is the precursor of all mRNA often called "premRNA", prior to the removal of introns.
Small nucleolar RNA (snoRNA) is found in the cell's nucleolus where it processes and
methylates rRNA.
Transfer RNA (tRNA) carries amino acids to nascent polypeptide chains synthesised on
the ribosomes.
DNA exists within our cells as chromosomes. Chromosomes are single moelcules
which contain regions that carry the information to produce or "encode" proteins or
RNA molecules. These regions are called genes and they are the most basic
functional genetic units in our chromosomes. We have approximately 35 000 genes
some of which are expressed contiuously ("house-keeping genes"), some only when
the cell is undergoing certain processes or only in cells that have matured in a
particular way, and some are expressed in response to an environmental stimulus.
The transcription start site defines a gene. Sequences "before" or 5' to the start site
are called upstream, and those after or 3' are called downstream. Pseudogenes
or remnants of duplicated genes that, due to mutation, no longer function are
sometimes found in humans.
When consisdering all of our DNA, including the genes and many other sequences
which do not encode proteins, we are talking about our genome. This name also
applies to viruses - although a viral genome has much less DNA (or RNA) than a
human genome.
A cistron is the smallest unit of DNA that can encode a protein. A cistron does not
include any regulatory or non-coding sequences.
Prokaryotic cells generally group their closely related genes and those genes
activated or inactivated at the same time, near to each other. The genes together
with their controlling elements are called operons and may be transcribed as a
single mRNA which is polycistronic, or capable of encoding several proteins.
Polycistronic messenger RNA (mRNA) consists of gene sequences separated by
intercistonic sequences. Preceding the first gene is a leader sequence and following
the last gene is a trailer sequence. The DNA between prokaryotic genes is called
intergenic DNA.
Eukaryotic cells organise their genome very differnetly. DNA encoding a gene's
precursor mRNA (pre-mRNA) is organised into regions called exons (EXpressed
sequences) which may be spread across thousands of nucleotide base pairs (bp). The
areas between exons ina gene are called introns (INtervening sequences).
Introns are not removed by luck, but with the aid of sequence specific splicing
signals. Most introns start (5') with the sequence GU and end (3') with an AG which
are referred to as the splice donor and splice acceptor sites. Another important
sequence is the branch site located 20-50 base pairs upstream (5') of the splice
acceptor site and containing a conserved A.
Five small nuclear RNA molecules (snRNA) and their proteins form a complex
called the spliceosome. When snRNA is associated with proteins they are known as
small nuclear ribonucleoproteins (snRNP; "snurps"). The five snRNPs which form
the spliceosome are called U1, U2, U4, U5 and U6. The splice donr site is attched to
the branch site to form a lasso or "lariat". Through an enzymatic process the intron
is then removed and the exons joined together.
As with many things in biology, there is more than one way for introns to be spliced.
Another form of intron removal involving a spliceosome is called alternative splicing
and is shown below. This relies upon alternative splice sites wihtin exons. This
process can produce more than one protein due to different ways of splicing the
same mRNA. Interestingly, eukaryotes carry a lot of DNA that does not appear to
encode any protein. This is often called junk DNA.
But intron removal can occur in the absence of a spliceosome, or in fact, any proteinbased enzyme at all. These introns are removed by self-splicing and rely upon the
action of catalytic RNA molecules called ribozymes. Self-splicing introns are divided
into two groups based on the way the chemoistry behind the splicing. Group I
introns are found in protozoa, fungal mitochondria, bacteriophage T4 and bacteria.
Group II introns exist in mitochondrial and chloroplast genes (plastids).
The region of mRNA that encodes the protein is called the coding sequence (cds)
and is a duplicate of the exon region of the DNA since the introns are removed from
the mRNA. Human genes are usually monocistronic meaning that each protein is
translated from a single mRNA.
Regulatory sequences on the DNA called enhancers, permit the binding of proteins
that control gene expression. Enhancer sequences may be kilobase pairs away from
the exons.
INTRODUCTION
The transmissible spongiform encephalopathies (TSEs) are a group of
invariably fatal neurodegenerative diseases found in a wide range of
mammals. The disease is found naturally in many ruminants (scrapie,
bovine spongiform encephalopathy-BSE), deer (chronic wasting
disease) and mink (transmissible mink encephalopathy), as well as
humans (see later). The disease can also be experimentally
transmitted to rodents, pigs and primates (6). TSEs are characterised
by long incubation times (in humans can be greater than 30 years),
and an infected individual will usually show some signs of progressive
ataxia, dysarthia, dysphagia, nystagmus, myoclonus and/or dementia.
The time from onset of symptoms to death is highly variable (in
humans it ranges from a few months to 10 years) (6).
THE INFECTIOUS AGENT
The TSEs are novel in that they are currently believed to be caused by an abnormal
folding of a host encoded protein, with no nucleic acid component (1), although this
hypothesis remains controversial. The protein has been named the prion protein
(PrP), the normal form of the protein is termed PrPC (Cellular), and the disease form
PrPSC (SCrapie). It is currently believed that the PrPSC form of the protein can arise
spontaneously, but that it can then go on to auto-catalyse the conversion of PrPC to
PrPSC (1).
The two forms of the protein have some different properties(2): PrPC is anchored to
the cell membrane by a glyco-phospho-inositol (GPI) anchor, whilst PrPSC
accumulates in endosomes; PrPSC accumulates in diseased individuals in plaque
deposits in the brain, and is partially resistant to proteolytic digestion with proteinase
K; and PrPSC is highly resistant to most sterilising procedures, and is not inactivated
by treatment with many sterilising agents such as UV light (3), nor by autoclaving
(4). However, the two proteins seem to have the same post-translational
modifications, and cannot be distinguished with monoclonal antibodies (5). In
addition, there are no in vitro assays which can be used to determine PrP SCbiological
activity. Much of the research in this area has therefore been concentrated on the
primary sequence of the PrP gene, as well as the use of transgenic animals carrying
different alleles of the gene.
THE PrP GENE
A schematic of the PrP gene is shown in Figure 1. The gene is c 750 base pairs (bp)
in length, coding for a c 250 amino acid (aa) protein, with the following domains
(7,8,9): the N-terminal 22 aas encode a cleaved signal peptide involved in transport
of the protein to the cell surface; the C-terminal 26 aas encode a signal sequence
that is cleaved in the golgi when the GPI anchor is added; there are two
glycosylation sites, and two C1 residues involved in intra-molecular disulfide bonding;
finally, there is a region in the N terminal half of the gene which encodes a series of
G-P1 rich octa peptide repeats. In humans, a number of pathogenic polymorphisms
have been described, which are responsible for the inherited forms of this disease
(see later).
Figure 1.
HUMAN DISEASE
There are 4 human diseases classified as TSEs. These are Creudtzfeldt-Jacob disease
(CJD), Gertsmann-Straussler syndrome (GSS), fatal familial insomnia (FFI) and kuru
(9). The latter is confined to the Fore tribe of Papua New Guinea (PNG), and is
caused by cannibalistic rituals, specifically the preparation and eating of human
brains. Since the widespread cessation of cannibalism in PNG, kuru has declined
dramatically, and is believed to have been wiped out. Cases which still arise are
thought to be due to the long incubation time of the disease, in people who engaged
in cannibalism earlier this century. CJD is the most common TSE diagnosed in
humans, and falls into three categories, iatrogenic, inherited, and sporadic.
Iatrogenic cases are extremely rare. They occur when contaminated material is
transplanted (eg cornea or dura mater transplants), or instruments used in neuroinvasive procedures are contaminated (eg depth electrodes). most of the cases are
due to batches of contaminated growth hormone prepared from human cadavers
(10,11). Sporadic CJD has an incidence rate of c 1/million people/year, world wide.
No correlation between sporadic CJD and populations that may be considered high
risk (eg abattoir workers, shepherds) has been observed. It is currently believed that
sporadic CJD arises through the spontaneous conversion of PrPCto PrPSC in an
individual (2,6). Inherited CJD is caused by pathogenic polymorphisms in the
human PrP gene. Such genetic lesions tend to be dominant although of variable
penetrance, although it is possible given the long incubation times of the disease
that asymptomatic people with a particular genetic lesion are dying of old age prior
to onset of disease. A large number of different polymorphisms have been described
for different lineages(12, 15). These include point mutations, such as p102l 1 (13), as
well as extra or fewer octa peptide repeats in the G-P rich region (12). GSS is an
inherited disease similar to inherited CJD. Indeed, the former can be considered a
sub-class of the latter.
In addition to pathogenic mutations, humans also have a neutral polymorphism,
M129V This mutation has been shown to have an effect on both sporadic and
inherited CJD. People who are M/M homozygous AND have the P102L mutation suffer
from FFI, whereas those who have M/V or V/V and the P102L mutation suffer from
typical CJD (14). FFI is characterised by progressive insomnia and torpor, with quite
different symptoms to typical CJD. Also, it has been shown that people who are
homozygous (M/M or V/V) are more likely susceptible to sporadic CJD than people
who are heterozygous at this allele (15).
THE BSE OUTBREAK
Changes in the processing of cow and sheep carcasses for rendering into bone meal
(protein supplement for cattle) in the UK in 1981/82 is the most likely cause of the
BSE epidemic (17). Solvent extraction and certain heating steps were removed, and
it is theorised that existing scrapie and BSE were no longer being inactivated, but
instead being passed back into the food chain (17). Whether BSE can or will transmit
to humans remains unknown at this time, with estimates of human infection ranging
from 0 to 10 million. Ten cases of "atypical" CJD have been described in the
literature (16). These cases were unusual in that they infected young people (median
age 32). The Spongiform Encephalopathy Advisory Committee (SEAC) said that
these cases were most likely caused by BSE, and on the strength of this the EC
banned all British beef imports (The UK exported 400 000 head of cattle in 1995).
However, much more research needs to be done before the transmission of BSE to
humans is proven.
Construction of a Lentivector
To construct a delivery vector, certain essential cis-acting sequences must be
retained within the retroviral vector genome. These include:
1.
2.
The packaging signal sequence (Y) which ensures the
encapsidation of the vector RNA into virions.
Elements that are necessary in the reverse transcription
process:



3.
Primer binding site (PBS) - which binds the tRNA primer
of reverse transcription
Terminal repeat (R) sequences - to guide reverse
transcriptase between the RNA strands during DNA synthesis
Purine-rich region 5' of the 3' LTR - which acts as the
priming site for synthesis of the second (+) DNA strand
Specific sequences near the ends of the LTRs that are
necessary for the integration of the proviral vector into the chromosome of
the host cell. The most common retroviral vector designs use the LTR of the
virus backbone and an internal promoter to drive the expression of the
foreign gene52. This approach gives rise to the phenomenon of "promoter
suppression". When selection is applied for one gene from multiple
transcription units, the expression of the other gene can be reduced or lost
completely.
An internal ribosome entry site (IRES) sequence may be used instead of an internal
promoter in a vector with two or more foreign genes to avoid the potential of
promoter suppression. An IRES permits multiple proteins to be produced from a
single vector without alternative splicing or multiple transcription units, hence
increasing the stability of the transferred gene53.
The essential minimum packaging signal (Y) sequence of lentiviruses is still unclear.
It is generally accepted to be in a region between the 5' LTR splice donor and the
gag start codon.
Addition of 5' gag sequences to the vector backbone has been shown to increase
packaging efficiency54,55. The 3' env gene fragment encompassing the Rev response
element (RRE) was reported to enhance the encapsidation of vectors when placed
upstream of the heterologous genes56,57.
Inclusion of this fragment allows accumulation of unspliced vector RNA in the
presence of Rev protein and enhances the transduction of recombinant vectors 56.
By incorporating the above information, the vector design, shown below, has the
potential to overcome the current low titer production and transduction efficiency of
lentiviral vectors.
Ideally, a recombinant lentiviral vector would be the best gene transfer system for
noncycling cells. It has promising clinical applications, especially for cystic fibrosis
gene therapy.
Lentivirus-based Vectors for CF Gene Therapy
Lentiviruses are part of the family Retroviridae. Like other retroviruses, they are
enveloped viruses that carry a core of RNA encoding their genetic information. The
structure of a lentivirus genome is shown in the figure below.
Figure 1. Schematic representation of lentiviral genome. LTR
(long terminal repeats) - contains viral promoter and enhancer.
SD - splicing donor. Y - packaging signal. Gag - codes for
virion core. Pol - generates reverse transcriptase,
endonuclease, and protease. Env - codes for envelope protein
Apart from boasting some of the best properties of current retrovirus vectors for
gene delivery (see previous section), lentiviruses are the only retroviruses able to
integrate into the chromosome of non-dividing cells. Gene transfer vectors based on
HIV-1 have been shown to transduce non-dividing cells effectively in vitro46.
Several research groups have described HIV-based vectors, unfortunately the virus
titers are extremely low47,48. Recently, a much higher production of vector stock was
achieved when an amphotropic envelope protein was substituted for the endogenous
HIV envelope protein in trans46. The amphotropic envelope protein, derived from
either murine leukemia virus (MLV) or vesticular stomatitis virus G (VSV-G),
broadened the tropism of the vector.
The use of the VSV-G envelope protein generates several problems. There is the
possible production of pseudovirions where other RNAs can be encapsidated instead
of recombinant virus49. The virus titer can be over-estimated as recombinant vectors,
empty vectors and pseudovirions can all be neutralized by anti-VSV antiserum.
There is no stable packaging cell line for VSV-G envelope proteins as these proteins
are cytotoxic50. The use of the MLV envelope protein generates no such problems.
However, in the reported experiment, the virus titer was fourfold lower than when
the VSV-G envelope was used.
Another hurdle to overcome in developing a novel recombinant lentiviral vector is the
current unavailability of a stable packaging cell line. Constitutive expression of
lentiviral proteins has been reported to be cytotoxic51. The use of inducible packaging
cells or a cell line with a high tolerance limit may overcome this problem.