Figure 1. Hepatitis C virus (HCV): model structure and genome organisation. (a) Model structure of HCV.
The left-hand side of the illustration shows the viral surface of envelope lipids and glycoproteins; the right-handside shows the RNA genome encased by capsid proteins. (b) Proteins encoded by the HCV genome.
HCV is formed by an enveloped particle harbouring a plus-strand RNA of ∼9.6 kb. The genome carries a long openreading frame (ORF) encoding a polyprotein precursor of 3010 amino acids. Translation of the HCV ORF is directed via a ∼340 nucleotide long 5' nontranslated region (NTR) functioning as an internal ribosome entrysite; it permits the direct binding of ribosomes in close proximity to the start codon of the ORF.
The HCVpolyprotein is cleaved co- and post-translationally by cellular and viral proteases into ten different products, with the structural proteins [core (C), E1 and E2] located in the N-terminal third and the nonstructural (NS2–5) replicative proteins in the remainder. Putative functions of the cleavage products are shown.
He Structure & Complexity of Virus Genomes
The composition & structure of virus genomes (i.e. the nucleic acid which encodes the genetic information of the virus) is more varied than any of those seen in the entire bacterial, plant or animal kingdoms. The nucleic acid comprising the genome may be single-stranded or double-stranded, & in a linear, circular or segmented configuration. Single-stranded virus genomes may be:
positive (+)sense, i.e. of the same polarity (nucleotide sequence) as mRNA
negative (-)sense
ambisense - a mixture of the two.
Virus genomes range in size from approximately 3,200 nucleotides (nt) (e.g. Hepadnaviruses) to approximately 1.2 million base pairs (Mbp, Mimivirus):
Unlike the genomes of all cells, which are composed of DNA, virus genomes may contain their genetic information encoded in either DNA or RNA.
Whatever the particular composition of a virus genome, all must conform to one condition. Since viruses are obligate intracellular parasites only able to replicate inside the appropriate host cells, the genome must contain information encoded in a form which can be recognised & decoded by the particular type of cell parasitized. Thus, the genetic code employed by the virus must match or at least be recognised by the host organism. Similarly, the control signals which direct the expression of virus genes must be appropriate to the host.
Many of the DNA viruses of eukaryotes closely resemble their host cells in terms of the biology of their genomes:
Some DNA virus genomes are complexed with cellular histones to form a chromatin-like structure inside the virus particle.
Vaccinia virus mRNAs were found to be polyadenylated at their 3' ends by Kates in 1970 - the first observation of this phenomenon.
Split genes containing non-coding introns, protein coding exons & spliced mRNAs were first discovered in adenoviruses by Sharp in 1977.
Molecular Genetics:
As already described, the new techniques of molecular biology have had a major influence in concentrating much attention on the virus genome. Initially, the questions to be asked about any virus genome will usually include the following:
Composition - DNA or RNA, single-stranded or double-stranded, linear or circular.
Size & number of segments.
Terminal structures.
Nucleotide sequence.
Coding capacity - open reading frames.
Regulatory signals - transcription enhancers, promoters & terminators.
Direct analysis by electron microscopy, if calibrated with known standards, can be used to estimate the size of nucleic acid molecules. The most important single technique has been gel electrophoresis. It is most common to use agarose gels to separate large nucleic acid molecules (several megabases or kilobases) & polyacrylamide gel electrophoresis (PAGE) to separate smaller pieces (a few hundred bp down to a few nucleotides). Nucleotide sequencing is dependent on the ability to separate molecules which differ from each other by only one nucleotide in size
The relative simplicity of virus genomes (compared with even the simplest cell) offers a major advantage - the ability to 'rescue' infectious virus from purified or cloned nucleic acids. Infection of cells caused by nucleic acid alone is referred to as transfection:
Virus genomes which consist of (+)sense RNA (i.e. the same polarity as mRNA) are infectious when the purified vRNA is applied to cells in the absence of any virus proteins. This is because (+)sense vRNA is essentially mRNA & the first event in a normally-infected cell is to translate the vRNA to make the virus proteins responsible for genome replication. In this case, direct introduction of RNA into cells merely circumvents the earliest stages of the replicative cycle.
In most cases, virus genomes which are composed of double-stranded DNA are also infectious. The events which occur here are a little more complex, since the virus genome must first be transcribed by host polymerases to produce mRNA. Using these techniques, virus can be rescued from cloned genomes, including those which have been manipulated in vitro.