Viral Genomes, Dr. Sobia Manzoor Molecular Virology Dr. Sobia Manzoor Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor VIRUS GENOMES A virion contains the genome of a virus in the form of one or more molecules of nucleic acid. For any one virus the genome is composed of either RNA or DNA. If a new virus is isolated, one way to determine whether it is an RNA virus or a DNA virus is to test its susceptibility to Ribonuclease (e.g., RNAse A, H, T) or deoxyribonuclease (e.g., DNAse 1, or S1 Nuclease). The virus nucleic acid will be susceptible to degradation by only one of these enzymes. Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor In order to optimize the cell for virus replication, Viruses also encode enzymes and proteins involved in modifying the cell in which the virus replicates. DNA Viruses utilize the infected cell’s nucleus as the site of genome replication share many common patterns of gene expression and genome replication along with similar processes occurring in the host cell. RNA Viruses have devised some way to replicate such since the cell does not have machinery for RNA-directed RNA replication. The replication of RNA viruses requires expression of specific enzymes that are not present in the uninfected host cell. Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor VIRUS GENOMES DNA genomes Examples ss, linear Parvoviruses ds, linear Poxviruses ss, circular Phage φX174 ds, circular SV40, Baculoviruses RNA genomes ss, linear Tobacco mosaic virus ds, linear Reoviruses ss, circular Hepatitis delta virus Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor GENOME SIZE Porcine circovirus (ssDNA) and hepatitis delta virus (ssRNA) each have a genome of about 1.7 kilobases (Kb). While at the other end of the scale there are viruses with dsDNA genomes comprised of over 1000 kilobase pairs (Kbp). The maximum size of the virus genome is subject to constraint are less severe for dsDNA all of the large virus genomes are composed of dsDNA. The largest RNA genomes known are those of some coronaviruses, which are 33kb of ssRNA. The largest virus genomes, such as that of the mimivirus, are larger than the smallest genomes of cellular organisms, such as some mycoplasmas. Virus genomes span a large range of sizes. Viral Genomes, Dr. Sobia Manzoor

GENOME MANIPULATION Mimivirus (1,181,404 bp) EsV-1 (335,593 bp) Human Cytomegalovirus (229,354 bp) Lambda (48,502 bp) φX174 (5375 b) MS2 (3569 b) Lambda (12 bp) Year 1971 1975 1977 1982 1990 2001 2004 The first genome sequenced The first DNA sequenced Viral Genomes, Dr. Sobia Manzoor

SECONDARY AND TERTIARY STRUCTURE The virus genome carries information, such as signals for the control of gene expression as well as encoding the virus protein (and in some cases untranslated RNAs) to be synthesized in the infected cell. Some of this information is contained within the nucleotide sequences, while for the single-stranded genomes some of it is contained within structures formed by intramolecular base pairing. Viral Genomes, Dr. Sobia Manzoor

SECONDARY AND TERTIARY STRUCTURE In ssDNA complementary sequences may base pair through G-C and A-T hydrogen bonding; in ssRNA weaker G-U bonds may form in addition to G-C and A-U base pairing. Intramolecular base pairing results in regions of secondary structure with stem loops and bulges. In some ssRNAs intramolecular base pairing results in structures known as pseudoknots Regions of secondary structures in single-stranded nucleic acids are folded into tertiary structures with specific shapes, many of which are important in molecular interactions during virus replication. The 5 end of poliovirus, HCV RNA, forms such structure called internal ribosome entry site (IRES) to which cell proteins bind to initiate translation. Some pseudoknots have enzyme activity, while others play a role in ribosomal frameshifting. Viral Genomes, Dr. Sobia Manzoor

A zinc finger in a protein molecule NON COVALENT PROTEIN DNA INTERACTIONS Proteins bound to viral nucleic acids are non-covalently attached. These proteins have regions that are rich in basic amino acids lysine and arginine which are positively charged and able to bind strongly to the negatively charged nucleic acids. DNA viruses such as Papillomaviruses and polyomaviruses have cell histones bound to the virus genome. Most proteins associated with virus genomes, however, are virus coded. Nucleic-acid-binding proteins may have other characteristics, such as zinc fingers the HIV-1 nucleocapsid protein has two zinc fingers. In some viruses such as tobacco mosaic virus the protein coating the genome constitutes the capsid of the virion. A zinc finger in a protein molecule A zinc finger has recurring cysteine and/or histidine residues at regular intervals. In this example there are two cysteines and two histidines. Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor SECONDARY STRUCTURES RESULTING FROM INTRAMOLECULAR BASE-PAIRING IN SINGLE-STRANDED NUCLEIC ACIDS. (a) Stem loops and bulges in ssRNA and ssDNA. (b) Formation of a pseodoknot in ssRNA. A pseudoknot is formed when a sequence in a loop (L1) base-pairs with a complementary sequence outside the loop. This forms a second loop (L2). Viral Genomes, Dr. Sobia Manzoor

MODIFICATIONS AT THE ENDS OF VIRUS GENOMES Examples ssRNA protein 5' 3' Poliovirus Cowpea mosaic virus An protein 5' 3' Barley yellow dwarf virus 5' SARS coronavirus An 3' 5' 3' Black beetle virus 5' 3' Cucumber mosaic virus tRNA-like structure 5' Rotaviruses virus 3' dsRNA 3' 5' protein 5' 3' Infectious pancreatic necrosis virus 3' 5' protein Viral Genomes, Dr. Sobia Manzoor

TERMINAL REPEATS IN VIRUS GENOMES Nucleic acid Type of repeat Examples XY dsDNA XY DTR Some herpes viruses, T phages XY XY XY dsDNA YX ITR Adenoviruses Tectiviruses (phages) YX XY XY ssDNA YX ITR Some Parvoviruses XY ssRNA(+) XY DTR Retroviruses ssRNA(-) YX ITR Influenza viruses, Bunyaviruses XY 1 DTR: direct terminal repeats ITR: inverted terminal repeats X and X represent complementary sequences. Y and Y represent complementary sequences. ssRNA (+) has the same sequence as the virus mRNA. ssRNA (-)has the sequence complementary to the virus mRNA. The RNAs of single-stranded RNA viruses with ITRs can circularize; a “panhandle” is formed by base pairing between the complementary sequences at the termini. yx yx Viral Genomes, Dr. Sobia Manzoor

STRATEGIES OF VIRAL m RNA SYNTHESIS FOR NUCLEUS-AND CYTOPLASM-BASED VIRUSES Site of mRNA synthesis Viral Genome (in Virion) Template for mRNA Synthesis Enzyme Responsible Examples Cytoplasm RNA Viral RdRP Picornaviruses(ss+),Reoviruses(ds), Rhabdoviruses (ss-) DNA Viral RNA polymerase Poxviruses (ds) Nucleus Host pol ll Polyomaviruses (ds),Parvoviruses(ss) Retroviruses(ss+) Viral RdRp Orthomyxoviruses(ss-) Hepatitis delta virus (ss-) Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor RNA MODIFICATIONS Capping Reovirus mRNA Methylated mRNA cap structure Splicing Ad2 mRNA Exons and introns Polyadenylation Poxvirus and SV40 mRNA Polyadenylation and signals for Polyadenylation RNA transport E1B55k,HVI-1Rev mRNA transport to cytoplasm Protein transport SV40 T antigen Nuclear localization signal (NLS) Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor Signal transduction V-rsc(RSV) Tyrosine kinases Protein modification Polyoma T antigen Tyrosine phosphorylation Malignant transformation Oncogenes (RSV), retroviral genome Proto-oncogenes, insertional mutagenesis Tumor suppressor genes SV40 T antigen,adenovirus E1A Papilloma virus E6/E7 Tumor suppressor genes (Rb, p53) Apoptosis Baculovirus IAP p35 Inhibitor of Apoptosis Proteins (IAP) Immune defenses Interferon Inactivated and live influenza virus Anti-viral state, viral interferon antagonists Adaptive immne response Lymphocytic choriomeningitis virus infection Antigen persentation and MHC restriction Viral Genomes, Dr. Sobia Manzoor

Viral Genomes, Dr. Sobia Manzoor CELLULAR TARGETS OF THE DNA TUMOR VIRUS ONCOPROTEINS Virus Gene Product Cellular Target Adenovirus E1A Rb E1B p53 SV40 Large T antigen Rb,p53 Polyomavirus Middle T antigen Src,Pl 3-k Papillomavirus E7 E6 E5 PDGF receptor Viral Genomes, Dr. Sobia Manzoor