VIRAL REPLICATIONViral replication is the term used by virologists to describe the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. From the perspective of the virus, the purpose of viral replication is to allow production and survival of its kind. By generating abundant copies of its genome and packaging these copies into viruses, the virus is able to continue infecting new hosts. Replication between viruses is greatly varied and depends on the type of genes involved. Viral populations do not grow through cell division, because they are acellular; instead, they use the machinery and metabolism of a host cell to produce multiple copies of themselves, and they assemble in the cell.The life cycle of viruses differs greatly between species but there are six basic stages in the life cycle of viruses: Attachment is a specific binding between viral capsid proteins and specific receptors on the host cellular surface. This specificity determines the host range of a virus. For example, HIV infects only human T cells, because its surface protein, gp120, can interact with CD4 and receptors on the T cell's surface. This mechanism has evolved to favour those viruses that only infect cells in which they are capable of replication. Attachment to the receptor can induce the viral-envelope protein to undergo changes that results in the fusion of viral and cellular membranes.

Penetration follows attachment; viruses enter the host cell through receptor mediated endocytosis or membrane fusion. This is often called viral entry. The infection of plant cells is different from that of animal cells. Plants have a rigid cell wall made of cellulose and viruses can only get inside the cells after trauma to the cell wall. Viruses such as tobacco mosaic virus can also move directly in plants, from cell to cell, through pores called plasmodesmata.[83] Bacteria, like plants, have strong cell walls that a virus must breach to infect the cell. Some viruses have evolved mechanisms that inject their genome into the bacterial cell while the viral capsid remains outside. Uncoating is a process in which the viral capsid is degraded by viral enzymes or host enzymes thus releasing the viral genomic nucleic acid.

Replication involves synthesis of viral messenger RNA (mRNA) for viruses except positive sense RNA viruses (see above), viral protein synthesis and assembly of viral proteins and viral genome replication.

Following the assembly of the virus particles, post-translational modification of the viral proteins often occurs. In viruses such as HIV, this modification (sometimes called maturation) occurs after the virus has been released from the host cell. Viruses are released from the host cell by lysisa process that kills the cell by bursting its membrane. Enveloped viruses (e.g., HIV) typically are released from the host cell by budding. During this process the virus acquires its envelope, which is a modified piece of the host's plasma membrane.

Protein synthesisProteins are essential to life. Cells produce new protein molecules from amino acid building blocks based on information coded in DNA. Each type of protein is a specialist that only performs one function, so if a cell needs to do something new, it must make a new protein. Viruses force the cell to make new proteins that the cell does not need, but are needed for the virus to reproduce. Protein synthesis basically consists of two major steps: transcription and translation.

Transcription is the process where information in DNA, called the genetic code, is used to produce RNA copies called messenger RNA (mRNA). These migrate through the cell and carry the code to ribosomes where it is used to make proteins. This is called translation because the protein's amino acid structure is determined by the mRNA's code.

Some RNA genes of viruses function directly as mRNA without further modification. For this reason, these viruses are called positive-sense RNA viruses. In other RNA viruses, the RNA is a complementary copy of mRNA and these viruses rely on the cell's or their own enzyme to make mRNA. These are called negative-sense RNA viruses. In viruses made from DNA, the method of mRNA production is similar to that of the cell. The species of viruses called retroviruses behave completely differently: they have RNA, but inside the host cell a DNA copy of their RNA is made. This DNA is then incorporated into the host's, and copied into mRNA by the cell's normal pathways. The genetic material within viruses, and the method by which the material is replicated, vary between different types of viruses.

DNA viruses

The genome replication of most DNA viruses takes place in the cell's nucleus. If the cell has the appropriate receptor on its surface, these viruses enter the cell by fusion with the cell membrane or by endocytosis. Most DNA viruses are entirely dependent on the host cell's DNA and RNA synthesising machinery, and RNA processing machinery. The viral genome must cross the cell's nuclear membrane to access this machinery. RNA viruses

These viruses are unique because their genetic information is encoded in RNA. Replication usually takes place in the cytoplasm. RNA viruses can be placed into about four different groups depending on their modes of replication. The polarity (whether or not it can be used directly to make proteins) of the RNA largely determines the replicative mechanism, and whether the genetic material is single-stranded or double-stranded. RNA viruses use their own RNA replicase enzymes to create copies of their genomes. Reverse transcribing viruses

These replicate using reverse transcription, which is the formation of DNA from an RNA template. Reverse transcribing viruses containing RNA genomes use a DNA intermediate to replicate, whereas those containing DNA genomes use an RNA intermediate during genome replication. Both types use the reverse transcriptase enzyme to carry out the nucleic acid conversion. Retroviruses often integrate the DNA produced by reverse transcription into the host genome. They are susceptible to antiviral drugs that inhibit the reverse transcriptase enzyme, e.g. zidovudine and lamivudine. An example of the first type is HIV, which is a retrovirus. Examples of the second type are the Hepadnaviridae, which includes Hepatitis B virus.

Baltimore Classification System

Viruses are classed into 7 types of genes, each of which have their own families of viruses, which in turn have differing replication strategies themselves. David Baltimore, a Nobel Prize-winning biologist, devised a system called the Baltimore Classification System to classify different viruses based on their unique replication strategy. There are seven different replication strategies based on this system (Baltimore Class I, II, III, IV, V, VI, VII). The seven classes of viruses are listed here briefly and in generalities.

Class 1: Double stranded DNA viruses

This type of virus usually must enter the host nucleus before it is able to replicate. Furthermore, these viruses require host cell polymerases to replicate its genome and hence is highly dependent on the cell cycle. Proper infection and production of progeny requires that the cell be in replication as that is when the cell's polymerases are active. The virus may induce the cell to forcefully undergo cell division, and chronically, this may lead to transformation of the cell and ultimately, cancer. An example of a family within this classification is the Adenoviridae.

Adenovirus genome consists of a linear double stranded DNA of about 36 kilobases pair; attached in a covalent linkage to 5 terminus of the DNA. This is a protein component essential for infectivity of DNA.Replication of viral DNA occurs in the nucleus.After the viral particle has been transported to the nucleus, the core is released and converted to DNA-Histone compex. Early transcription is done by RNA polymerase of the host and a number of primary transcripts are made.The transcripts are spliced, capped and equally polyadenylated to give several different mRNAs. Early proteins are involved in the regulation of DNA replication, later proteins are the virus coat proteins. Viral DNA replication uses viral encoded proteins as a primer and another virus encoded protein as DNA polymerase. Replication of a linear dsDNA molecule e.g. adenovins, the imitation can begin at either end or at both ends simultaneously.Two strands are produced which are rounded/double single strands. They later cyclize by means of the inverted terminal repeats and a new complimentary strand is synthesized beginning from the 5 end. The product being another double stranded molecule. This mechanism of replication is interesting because it does not involve the formation of discontinuous fragments of DNA of the lagging strand as occurs in conventional DNA replication.

There is only one well studied example in which a class 1 virus is not replicating within the nucleus, that is the Poxvirus family, a highly pathogenic virus that infects vertebrates. The example is a smallpox virus.

Class 2: Single stranded DNA viruses

Viruses that fall under this category includes ones that are not as well studied, but still do pertain highly to vertebrates. Two examples include the Circoviridae and Parvoviridae. They replicate within the nucleus, and form a double stranded DNA intermediate during replication. A human Circovirus called TTV is included within this classification and is found in most all humans, infecting them asymptomatically in nearly every major organ.

Class 3: Double stranded RNA viruses

Like most viruses with RNA genomes, double stranded RNA viruses do not rely on host polymerases for replication to extent that viruses with DNA genomes do. Double stranded RNA viruses are not as well studied as other classes. This class includes two major families, the Reoviridae and Birnaviridae. Replication is monocistronic and includes individual, segmented genomes, meaning that each of the genes code for only one protein, unlike other viruses which exhibit more complex translation.

Class 4 & 5: Single stranded RNA viruses

These viruses consist of two types, however both share the fact that replication is primarily in the cytoplasm, and that replication is not as dependent on the cell cycle as DNA viruses. This class of viruses are also one of the most studied types of viruses, alongside the double stranded DNA viruses.

Class 4: Single stranded RNA viruses - Positive (+) sense

The positive sense RNA viruses and indeed all genes defined as positive sense can be directly accessed by host ribosomes to immediately form proteins. These can be divided into two groups, both of which reproduce in the cytoplasm:

Viruses with polycistronic mRNA where the genome RNA forms the mRNA and is translated into a polyprotein product that is subsequently cleaved to form the mature proteins. This means that the gene can utilize a few methods in which to produce proteins from the same strand of RNA, all in the sake of reducing the size of its gene.

Viruses with complex transcription, for which subgenomic mRNAs, ribosomal frameshifting and proteolytic processing of polyproteins may be used. All of which are different mechanisms with which to produce proteins from the same strand of RNA.

Examples of this class include the families Coronaviridae, Flaviviridae and Picornaviridae.Class 5: Single stranded RNA viruses - Negative (-) sense

The negative sense RNA viruses and indeed all genes defined as negative sense cannot be directly accessed by host polymerases to immediately form proteins. Instead, they must be transcribed by viral polymerases into a "readable" form, which is the positive sense reciprocal. These can also be divided into two groups:

Viruses containing non segmented genomes for which the first step in replication is transcription from the (-) stranded genome by the viral RNA-dependent RNA polymerase to yield monocistronic mRNAs that code for the various viral proteins. A (+) sense genome copy is then produced that serves as template for production of the (-) strand genome. Replication is within the cytoplasm.

Viruses with segmented genomes for which replication occurs in the nucleus and for which the viral RNA-dependent RNA polymerase produces monocistronic mRNAs from each genome segment. The largest difference between the two is the location of replication.

Examples in this class include the families Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Filoviridae and Rhabdoviridae (which includes rabies).The negative RNA is transcribed in the cytoplasm of the cell into distinct RNA namely;

1.+ssRNA which acts as mRNA.

2.+ssRNA which is complimentary of the viral genome.

The +ssRNA is used as a template for the synthesis of -ssRNA( viral genome) mRNA may be monocistronic coding for only one protein.The poly A-tail is not at the end of mRNA and serves as stop codon.The synthesis of RNA polymerase which initiates the formation of many +ss sRNA is also in place. The transcription of viral mRNA leads to synthesis of coat protein and copying the full length +ssRNA leads to the formation of full length ssRNA, Assembly follows, where the nucleocapsid proteins and the capsid envelope proteins are brought together. The nucleic acid(RNA) of the orthomyxovirus exists in different fragments in the capsid i.e. exists a segmented genome e.g. influenza virus has 8 fragments or segments of RNA in the capsid.Class 6: Positive (+) sense single stranded RNA viruses that replicate through a DNA intermediate

A well studied family of this class of viruses include the retroviruses. One defining feature is the use of reverse transcriptase to convert the positive sense RNA into DNA. Instead of using the RNA for templates of proteins, they use DNA to create the templates, which is spliced into the host genome using integrase. Replication can then commence with the help of the host cell's polymerases. A well studied example includes HIV.RNA is converted dsDNA which is a linear molecule by the enzyme reverse transcription in the cytoplasm. Reverse transcription enzyme is essentially a DNA polymerase with three enzymatic activities.

1.synthesis of DNA with a DNA template.2.Synthesis of DNA with a RNA template.

3.Ribonuclease-H activity (it degrades the RNA strand of a RNA-DNA hybrid)

A primer is required by the two enzymes,reverse tanscription needs a primer for DNA synthesis which is a specific cellular transfer RNA (+RNA). Using the RNA primer the 100 or so nucleoids at the 5 terminus of the RNA are reversed transcribed into DNA. Once transcription reaches the 5end of RNA, the transcription process stops. The ribonuclease H activity leads to formation of a small, single stranded DNA that is complimentary to the RNA segment at the other end of the viral DNA. This small ssDNA hybrid with the other end of the viral RNA molecule,while the copying molecule of the viral RNA sequence is continued. The continued action of the reverse transcriptase and ribonuclease H leads to formation of dsDNA molecule with long terminal repeats at each end. The lts contain strong promoters of transcription and are involved in the integration process. A provirus is a stable genetic element after a succesful intergration of the viral DNA into the host DNA.Class 7: Double stranded DNA viruses that replicate through a single stranded RNA intermediate

This small group of viruses, exemplified by the Hepatitis B virus, have a double-stranded, gapped genome that is subsequently filled in to form a covalently closed circle (ccc DNA) that serves as a template for production of viral mRNAs and a subgenomic RNA. The pregenome RNA serves as template for the viral reverse transcriptase and for production of the DNA genome.SUMMARY

I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) II: Single-stranded (+)sense DNA (Parvoviruses) III: Double-stranded RNA (Reoviruses; Birnaviruses) IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc) V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc) VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses) VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses)

REFERENCE: 88I.N.J. Dimmock et al. "Introduction to Modern Virology, 6th edition." Blackwell Publishing, 2007.SMB 301: INTRO TO VIROLOGY KU-MAIN CAMPUS BSc. MICROBIOLOGY

Lecturer: Shem Peter Mutua Mutuirismutuiri@gmail.com