Virus Genome Vi G & Virus Replication

Mukund S. Vats

Genome
virus the genome is composed of either RNA or DNA How to determine whether it is an RNA virus or a DNA virus?

 Enzyme Based Identification . Ribonuclease ( ib l (RNAase) ) Deoxyribonuclease (DNAase)

 Four types: yp

1. dsDNA, 2. ssDNA, 3. dsRNA 4. ssRNA. Two Types 1. Circular 2. Linear

Genome size
large range of sizes May vary between 1.7 kb to over 1000 kb The l h largest virus genomes, such as that of the i h h f h mimivirus, are larger than the smallest genomes of cellular organisms, such as some f ll l i h mycoplasmas

Genome Contents
Encodes the virus proteins Some cases untranslated RNAs The virus genome carries additional information, such as
Signals for the control of gene expression. Signals for down regulations of expressed proteins. Signals for viral protein processing.

Generally by nucleotide sequences, cases, genomes, In some cases mostly with single stranded genomes within structure intra molecular base pairing i.e. secondary and tertiary structures have been observed to regulate the expression expression.

Secondary and tertiary structure

In single stranded Nucleic Acids. WHY? ssDNA complementary sequences may base pair through GC and AT hydrogen bonding; ssRNA weaker GU bonds may form in addition to GC and AU base pairing. Intramolecular b I t l l base pairing ii results in regions of secondary structure with stemloops and g bulges. In some ssRNAs intramolecular base pairing results in structures known as pseudoknots

Escaping The Cell Defense.

End E d capping / Tagging .. i T i

 In addition to this the genomes of viruses are base paired and folded near their 3 ends to form structures similar to transfer RNA RNA. These structures contain sequences that promote the initiation of RNA synthesis synthesis.

Repeat Sequences
The genomes of many viruses contain sequences that are repeated repeated. These sequences include promoters, origins of replication and other elements that are involved in the control of events in virus replication. l Many linear virus genomes have repeat sequences at the ends (termini), Known as p Terminal Repeats If the repeats are in the same orientation they are known as direct terminal repeats (DTRs), whereas if they are in the opposite orientation they are known as inverted terminal repeats (ITRs). Multiple repeated copies of a gene present in some virus DNAs Known as tandem repeats

Virus Replication
(making of new Replica of virus

What is difference between Virus replication & genome replication?

Overview Of Virus Replication

1. Attachment of a virion to a cell 2. Entry and establishment into the cell 3. Transcription of virus genes into messenger RNA molecules (mRNAs) 4. Translation of virus mRNAs into virus proteins 5. 5 Genome replication 6. Assembly of proteins and genomes into virions 7. 7 Exit of the virions from the cell cell.

1. Attachment of a virion to a cell

Via Cell receptors and / or co receptors usually glycoprotein with a wide range of functions
acting as receptors for chemokines and growth factors; mediating cell to cell contact and adhesion

Addition to cell receptor cascade of reactions Conformational Change in virus receptor attachment to Co receptor inside the cell cell.

Evidence that a cell surface molecule is a virus receptor If we block or develop that particular Receptor
Soluble derivatives of the molecule block virus binding/infectivity. binding/infectivity The normal ligand for the molecule blocks virus binding/infectivity. Introduction of the gene encoding the molecule into virus resistant cells, and expression of that , p gene, makes those cells susceptible to infection.

Attachment of virions to receptors

Receptor binding by hydrogen bonds ionic bonds, attractions and vander Waal forces. No covalent bonds are formed between virions and receptors I is a weak b d b It i k bond between. why? h ?
To reduce chances of dual infection. Th receptor configuration may change easily. The fi i h il

2. 2 Entry into the cell

After binding to receptors viruses must cross the g p plasma membrane.
They may either fuse to cell surface or They may cross the membrane through endocytosis into the cytoplasm.

This process is used by cells for a variety of p y y functions, including nutrient uptake and defense against pathogens. Two common processes (may iit b iindependent off these two) be d d h
Clathrin mediated endocytosis (e.g. adenoviruses) Caveolin mediated endocytosis (e.g. simian virus 40)

Change in conformation may be because of: Binding Of Receptor Change in PH

Carried to lysosomes where the pH may be further lowered by a process that pumps hydrogen ions across the membrane This acidification of the environment of the virion is important for those enveloped viruses that need to carry out acid triggered fusion of the envelope with y gg p the vesicle membrane. Lipid bi layers do not fuse spontaneously and each enveloped virus has a specialized glycoprotein responsible for membrane fusion.

Naked Viruses

Enveloped Viruses

Intracellular transport
Most of DNA viruses the destination of nucleus is reached using one of the transport systems of the cell, such as the microtubules. Most M t RNA viruses of eukaryotes replicate i th cytoplasm & M j it of th i f k t li t in the t l Majority f them encode all the enzymes for replication of their genomes and they have no requirement for the enzymes from nucleus. Retroviruses are RNA viruses, replicate their genomes in the nucleus. Then they it in the t l Th th wait i th cytoplasm until mitosis b i til it i begins. During mitosis the nuclear envelope is temporarily broken down and the virus DNA (with associated proteins) is able to enter the nuclear compartment. These viruses therefore can replicate only in cells that are dividing. However DNA (with associated proteins) of a group of retroviruses, however, can be transported into an intact nucleus. This group (the lentiviruses, which includes HIV) can therefore replicate in non dividing but active cells.

dsDNA dsRNA ssRNA (+) ssRNA ( )

dsDNA ssDNA

Generaly DNA viruses replicate in the nucleus (Except few ds DNA viruses). The structural proteins of some of these viruses have sequences that allow them to attach to microtubules. A number of viruses uses this transport system to take their nucleo capsid, from the periphery of the cytoplasm to a location close to the nucleus. Viruses must either shed some of their load to form slimmer structures or un coat at a nuclear pore. Depending on the virus, the process can take place
at the cell surface, the capsid remaining on the exterior surface of the cell; within the cytoplasm; at a nuclear pore; within the nucleus.

3 a sc pt o of us genes to 3. Transcription o virus ge es into mRNAs s

Central Dogma and modification

1958 Francis Crick Proposed Central Dogma, which is modified in 1970

gene transcription
viruses transcribe their genes by similar processes, some of them using parts of the cell transcription machinery. Hence, the expression of viral l gene is also l controlled by various sequences in the DNA: q
binding sites for transcription factors, which affect the rate of transcription

Enhancers

Promoter T A T A A/T A A/T A/G terminator

 Viruses that replicate in the nucleus generally use a cell g y enzyme, while viruses that replicate in the cytoplasm encode their own. An RNA virus in cytoplasm needs an RNA dependent RNA polymerase to transcribe. Each virus in Classes III, IV and V encodes its own enzyme, in spite of the fact that the cells of plants and some other eukaryotes encode ssRNA dependent RNA polymerases.

 All the viruses that carry out transcription in the cytoplasm have the transcriptase in the cytoplasm, virion. (except the (+) RNA viruses) Before the (+) RNA viruses can begin transcription they must translate copies of the enzyme from the genome RNA. Among the synthesized mRNA most transcripts are capped 5 end or have poly A capped 5 tail at the 3 end.

 Splicing of mRNA Some primary transcripts of viruses that replicate in the nucleus are processed i the same way to produce the virus mRNAs d in h d h i RNA The HIV 1 genome has a number of splice donor sites and acceptor sites; splicing of the primary transcript results in more than 30 different mRNA species

4. Translation of virus mRNAs into virus proteins p

Eukaryotic mRNA is monocystronic. methylated nucleotide cap at the 5 end and a poly(A) sequence at the 3 end play key roles in the initiation of translation Cap p binding site for g eukaryotic initiation factors (eIFs), Poly(A) tail poly(A) tail binding protein binds to it which further bound at the ends of the RNA and are able to interact and stimulates translation.

Translation from bicistronic mRNAs

HIV Vpu and Env

Rotavirus NSP5 and NSP6

HIV 1 Gag and Gag Pol

There are a number of virus mRNAs that h h have two or more ORFs In many bicistronic mRNAs the ORFs th ORF overlap; i others l in th there is an ORF within an ORF Frame shifting occ rs when occurs hen the ribosome moving along the RNA encounters a frame shift signal (a specific sequence) followed by a secondary structure, usually a pseudoknot

post translational modification of proteins

Once protein synthesized may undergo one or O ce p ote sy t es ed ay u de go o e o more modifications, including y y Addition of Glycosylation in RER oligosaccharide groups to the polypeptide chain, Acylation Addition of an acyl group (RCO) to a molecule facilitates attachment to plasma membrane. Ph h l ti Phosphorylation involves th t i l the transfer of a f f phosphate group from a nucleotide by protein kinase important for proper configuration configuration.

Bacterial Translation & Eukaryotic Translation

Bacterial translation differs from eukaryotic translation in a number of features.

Translation may start before transcription is complete. The lack of a nucleus allows transcription and translation to be coupled. The ribosomes are smaller (the ribosomal subunits have sedimentation coefficients of 30S and 50S). The 30S ribosomal subunit binds directly to a translation initiation region on the mRNA Initiation generally involves interaction of the Shine Dalgarno (S D) mRNA. sequence at the ribosome binding site (RBS) on mRNA and the anti S D sequence at the 3 end of 16S rRNA in the 30S subunit. The methionine of the initiator methionyl tRNA is generally formylated. y g y y A much smaller number of initiation factors areinvolved. All ORFs within an mRNA are translated and several may be translated concurrently. A few phages have overlapping genes, which may be translated by reading through a stop codon or by ribosomal frame shifting

Transport of proteins
Virus molecules synthesized in the infected cell must also be transported. t t d Virus mRNAs are transported from the nucleus to the cytoplasm, and virus proteins may be transported to various locations, including the nucleus Protein synthesis begins on a free ribosome, but when the signal sequence has been synthesized it directs the polypeptideribosome complex to the endoplasmic , p y reticulum, where protein synthesis continues. Many proteins have a sequence of amino acids (post code) that specifies their destination destination.

Many of the proteins synthesized in the rough endoplasmic reticulum are transported via vesicles to the Golgi complex From here the glycoproteins may be transported to other membranes, such as the plasma membrane or the nuclear envelope. Progeny virions may bud from these membranes Nuclear genome replication of virus requires various viral proteins to be transported back to nucleus. These proteins have a nuclear localization signal which drive them to nucleus. RNAs are also transported within the cell l d h h ll mRNAs synthesized in the nucleus must be exported through nuclear pores to the cytoplasm. The RNAs are taken to their destinations by p proteins. The Rev protein of HIV 1 has both a nuclear localization signal and a nuclear export signal. The nuclear localization signal ensures that Rev is transported into the nucleus, nucleus where it binds specifically to HIV 1 RNA The nuclear export signal RNA. ensures that Rev and its RNA cargo are transported from the nucleus to the cytoplasm via a nuclear pore.