Replication of viruses 5 Dr Amanj Saeed MB.CH.B, MSc, PhD 2016

Nucleotide chain Individual Nucleotides P CH2 Base P CH2 Base P CH2 5 P CH2 1 Base Individual Nucleotides 4 Sugar 3 2 5 P CH2 1 Base 4 Sugar 3 2 5 P CH2 1 Base 4 Sugar 3 2

Polynucleotide chain 5’ 3’ 3’ 5’

ATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAG MAGRSGDSDEELIRTVRLIKLLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILGTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTVLESGTKE

RNA Sense In virology, the genome of an RNA virus can be said to be either positive-sense, also known as a "plus-strand", or negative-sense, also known as a "minus-strand“. Whether a virus genome is positive-sense or negative-sense can be used as a basis for classifying viruses.

RNA Sense Positive-sense RNA Positive-sense (5' to 3') viral RNA signifies that a particular viral RNA sequence may be directly translated into the desired viral proteins. In positive-sense RNA viruses, the viral RNA genome can be considered viral mRNA, and can be immediately translated by the host cell. Some viruses like Coronaviridae have positive-sense genomes that can act as mRNA and be used directly to synthesize proteins without the help of a complementary RNA intermediate. these viruses do not need to have an RNA polymerase packaged into the virion.

RNA Sense Negative-sense Negative-sense (3' to 5') viral RNA is complementary to the viral mRNA and thus must be converted to positive-sense RNA by an RNA-plymerase prior to translation. Negative-sense RNA has a nucleotide sequence complementary to the mRNA that it encodes. This RNA cannot be translated into protein directly. Instead, it must first be transcribed into a positive-sense RNA that acts as an mRNA. Some viruses (like Influenza) have negative-sense genomes and so must carry an RNA_polymerase inside the Virion

Conformation of viral genomes RNA viruses DNA viruses Double stranded genome Or single stranded (linear, circular, continuous, segmented) The nucleic acid of all DNA viruses is double stranded (Except parvovirus) and Hepadnavirus. The nucleic acid of all RNA viruses is single stranded (except reoviruses )

Viral genome size Human cells have more than 60 000 genes E-coli has 4000 genes Largest virus like (Poxviruses) may contain only 200 or fewer genes Smallest viruses may have 3 or 4 genes RNA viruses have smaller genomes and codes for fewer proteins than DNA viruses

Viral genome size Viral genome can be measured in terms of bases (or nucleotide in their nucleic acid. These Numbers are expressed as thousands of bases (Kilobases , or kb) for single stranded genomes the notation (kb) is used . For double stranded genomes the notation kilobase pair (kbp) is used.

Viral genome size Example: Single stranded measles virus genome is 16 000 bp (16 kb) Double stranded adenovirus genome is 36 000 bp (36 kbp)

Viral replication Virus infection depend on chance contact (Virus should enter at suitable site and in large numbers) Infection will follow by transcription of incoming viral genes to form viral mRNA, This is followed by translation of mRNA to produce early viral proteins (enzymes necessary to replicate viral DNA or RNA).

What information encoded in a viral genome ? Gene products and regulatory signals for: Replication of the viral genome Assembly and packing of the genome. Regulation and timing of the replication cycle . Modulation of the host defense. Spread to other host cells.

Information not contained in viral genome No genes encoding the complete protein synthesis machinery. No genes encoding proteins involved in energy production or membrane biosynthesis. No classical centromere or telomere. 90% of giant viral genes are novel

Early viral proteins Transcription of Viral DNA to produce various viral mRNAs, which code for early viral proteins. Early viral proteins may have various function, Like: DNA dependent DNA polymerase that catalyse and direct the synthesis of new viral DNA molecule. Others are transcription activators

Late viral proteins Late viral mRNA are transcribed only from newly synthesised viral DNA. Late viral proteins are viral structural proteins. Virus structural proteins with new DNA to form progeny virion. New virions are released by mixture of budding and cell lysis.

Strategies for Viral replication Recognition of target host cells: All viruses have a receptor binding protein on their surface that react with corresponding receptor on a cell surface. These receptor have other functions and virus simply use them for attachment. Viral receptors are often glycoproteins or glycolipids. Viruses are restricted to given host and, within that host, to particular cell and tissue.

Strategies for Viral replication Example: HIV specifically interact with two receptors on certain T lymphocyte and other cells. Primary receptor is CD4 molecule Secondary receptor is chemokine receptor molecule (CXCR-4) or a β-chemokine receptor molecule.

Strategies for Viral replication internalisation of viruses After attachment virus must penetrate the external plasma membrane of the cell and release its genome in to the cell. Three ways of internalisation: Fusion from without: fusion at the cellular plasma membrane (Measles, Mumps and HIV). these viruses have a fusion protein that mediate fusion between the lipid of the virus and the lipids of cell membrane.

Strategies for Viral replication Receptor Mediated endocytosis (viropexis) this is the most common entry technique for viruses . Mammalian cells developed methods for attachment and entry of nutrient and hormones, viruses can use those methods for entry. after attachment of the virus inversion of the cellular membrane and associated virus occurs. after that the virus is in the cytoplasm but bounded by cell membrane (endosome) the virus will be released from endosome by internal fusion .

Strategies for Viral replication C. Clathrin and caveolin independent pathway

Strategies for Viral replication 3. Formation of mRNA Positive-stranded parental viral RNA with the addition of poly (A) (AAA) tract at the 3’ end and a cap at 5’ end is used directly as viral mRNA from which early and late viral proteins are translated directly. (eg, polio and flaviviruses) In negative stranded RNA viruses, viral RNA polymerase must first create mirror image copies of the original negative strand viral RNA segments. These copies are now positive sense, and function as viral mRNA which will be translated to viral proteins. Example Influenza and Rabies viruses.

Double stranded DNA

Single stranded DNA

Double stranded RNA

Single stranded RNA

Retroviruses

Negative sense single stranded RNA

Attachment to cell receptors Infecting virus Attachment to cell receptors Penetration Un-coating Replication Assembly Release Lytic virus (no envelop) Budding virus (envelope) Synthesis of viral mRNA Synthesis of viral proteins Synthesis of viral nucleic acid

Strategies for Viral replication Retroviruses They have more complicated strategy for producing viral mRNA. When the virus infect a cell the parental viral RNA is reverse transcribed by viral reverse transcriptase (RT) which converts the viral RNA genome to DNA –RNA hybrid. The RNA strand is digested away from the hybrid and replaced by DNA copy to give rise to dsDNA molecule.

Strategies for Viral replication Retroviruses The dsDNA integrates in to chromosomal DNA by viral enzyme (integrase). At this stage the integrated viral DNA is called pro-viral DNA. Viral mRNA are transcribed from proviral DNA in the same way as host mRNA. The viral messages are translated and viral proteins are syntesized.

HIV lifecycle virus binding fusion ssRNA (+) reverse transcription dsDNA nuclear transport integration nucleus cytoplasm transcription virion assembly and release translation of viral proteins maturation HIV lifecycle

Strategies for Viral replication DNA viruses DNA viruses will also produce mRNA transcripts after the infection of a cell. This done by host cell enzyme (DNA dependent RNA polymerase) Some viruses like poxvirus carry the enzyme. Some times early and late viral mRNA transcribed from dsDNA. Some viruses early mRNA transcribed from input parental DNA, while late mRNA transcribed from newly replicated viral DNA

Strategies for Viral replication DNA Viruses All DNA viruses replicate their genome in cell Nucleus (Except Poxviruses). Replication of DNA virus genome depend on the configuration of DNA: linear and single stranded DNA Circular DNA Linear and double stranded Replication of single stranded DNA involves the formation of double stranded intermediate which serves as a template for the synthesis of single stranded progeny DNA.

Synthesis of viral proteins All viruses uses the cellular ribosome to translate viral mRNA. Viral messages are translated in to: structural proteins that constitutes the viral particle . Non-structural proteins which are enzymes or transcription factors for virus replication. Viruses use overlapping reading frame strategy (two or more polypeptide can be coded in a single length of nucleic acid.

Synthesis of viral proteins Post translation modification of viral proteins: viral proteins must fold correctly into a precise three-dimensional structure. Post translation modification is required to initiate folding. glycosylation. covalent attachment of lipoic acid. Addition of phosphate, sulphate, and acyl group.

Virus assembly and release After viral replication and synthesis of structural and non-structural proteins, it is time for assembly. Some viruses assemble in cytoplasm and others predominantly assemble in nucleus. Most enveloped viruses bud through plasma membrane, but few exploit the endoplasmic reticulum membrane. Some viruses (like polio virus) are released on lysis and death of the cell (known as lytic viruses). Other viruses like (HIV) escape by budding from the cell surfaces

Virus assembly and release In case of budding viruses, the synthesised viral proteins are transported to the plasma membrane, the structural proteins migrate to the inside of plasma membrane . The protein and nucleic acid assemble and viral DNA or RNA is packaged. Finally completed virion buds by protrusion through plasma membrane. Some viruses like HIV and influenza undergo post-release maturation.