Lecture 3 Genomes and Genetics.pdf

3 Genomes and Genetics Introduction The “Big and Small” of Viral Genomes: Does Size Matter? Genome Principles and the Bal­ti­more System The Origin of Viral Genomes Structure and Complexity...

3 Genomes and Genetics Introduction The “Big and Small” of Viral Genomes: Does Size Matter? Genome Principles and the Bal­ti­more System The Origin of Viral Genomes Structure and Complexity of Viral Genetic Analysis of Viruses Genomes Classical Genetic Methods DNA Genomes Engineering Mutations into Viral RNA Genomes Genomes What Do Viral Genomes Look Engineering Viral Genomes: Viral Vectors Like? Perspectives Coding Strategies References What Can Viral Sequences Tell Us? Study Questions LINKS FOR CHAPTER 3 Virocentricity with Eu­gene Koonin CRISPR-Cas im­mune sys­tems http:// ​bit.​ly/​Virology_​Twiv275 http://​microbe.​t v/​t wim/​t wim184  pragmatically, the system simplifies comprehension of the ex­ traordinary reproduction cycles of viruses. The Baltimore system omits the second universal function of viral genomes, to serve as a template for synthesis of prog­ eny genomes. Nevertheless, there is also a finite number of nucleic acid­copying strategies, each with unique primer, Introduction template, and termination requirements. We shall combine Earth abounds with uncountable numbers of viruses of great this principle with that embodied in the Baltimore system to diversity. However, because taxonomists have devised meth­ define seven strategies based on mRNA synthesis and ge­ ods of classify ing viruses, the number of identifiable groups nome replication. The Baltimore system has stood the test of is manageable (Chapter 1). One of the contributions of molec­ time: despite the discovery of multitudes of viral genome se­ ular biology has been a detailed analysis of the genetic mate­ quences, they all fall into one of the seven classes. rial of representatives of major virus fami lies. From these Replication and mRNA synthesis present no obvious chal­ studies emerged the principle that the viral genome is the nu­ lenges for most viruses with DNA genomes, as all cells use cleic acid­based repository of the information needed to DNA­based mechanisms. In contrast, animal cells possess no build, reproduce, and transmit a virus (Box 3.1). These ana ly­ known systems to copy viral RNA templates and to produce ses also revealed that the thousands of distinct viruses defined mRNA from them. For RNA viruses to propagate, their RNA by classical taxonomic methods can be organized into seven genomes must, by definition, encode a nucleic acid polymerase. groups, based on the structures of their genomes. Structure and Complexity Genome Principles and the of Viral Genomes Baltimore System Despite the simplicity of expression strategies, the composition A universal function of viral genomes is to specify proteins. and structures of viral genomes are far more varied than those However, none of these genomes encode the complete ma­ seen in the entire archaeal, bacterial, or eukaryotic domains. chinery needed to carry out protein synthesis. Consequently, Nearly every possible method for encoding information in nu­ one important principle is that all viral genomes must be cop­ cleic acid can be found in viruses. Viral genomes can be ied to produce messenger RNAs (mRNAs) that can be read by host ribosomes. Literally, all viruses are parasites of their host DNA or RNA cells’ translation system. DNA with short segments of RNA A second principle is that there is unity in diversity: evolu­ DNA or RNA with cova lently attached protein tion has led to the formation of only seven major types of vi­ single­stranded (+) strand, (−) strand, or ambisense ral genome. The Baltimore classification system integrates (Box 3.2) these two principles to construct an elegant molecular algo­ double stranded rithm for virologists (Fig. 3.1). When the bewildering array of linear viruses is classified by this system, we find seven pathways to mRNA. The value of the Baltimore system is that by know ing circular only the nature of the viral genome, one can deduce the basic segmented steps that must take place to produce mRNA. Perhaps more gapped P R I N C I P L E S Genomes and Genetics The genomes of viruses range from the extraordinarily small Although the details of replication difer, all viruses with (2,500 kbp); the diver­ RNA genomes must encode either an RNA­dependent sity in size likely provides advantages in the niches in which RNA polymerase to synthesize RNA from an RNA template particular viruses exist. or a reverse transcriptase to convert viral RNA to DNA. Viral genomes specify some, but never all, of the proteins The information encoded in viral genomes is optimized by a needed to complete the viral reproductive cycle. variety of mechanisms; the smaller the genome, the greater the compression of genetic information. That only seven viral genome replication strategies exist for all known viruses implies unity in viral diversity. The genome sequence of a virus is at best a biological “parts list” and tells us little about how the virus interacts with its host. Some genomes can enter the reproduction cycle upon en­ try into a target cell, whereas others require prior repair Technical advances allowing the introduction of mutations or synthesis of viral gene products before replication can into any viral gene or genome sequence are responsible for proceed. much of what we know about viruses. 63 64 Chapter 3 B OX 3.1 B OX 3.2 B A C K G R O U N D T E R M I N O L O G Y What in­for­ma­tion is en­coded in a vi­ral Important con­ven­tions: plus (+) and mi­nus (−) ge­nome? strands Gene prod­ucts and reg­u­la­tory sig­nals re­quired for mRNA is de­fined as the pos­i­t ive (+) strand, be­cause it can be trans­ lated. A strand of DNA of the equiv­a ­lent po­lar­ity is also des­ig­nated rep­li­ca­tion of the ge­nome as a (+) strand; i.e., if it were mRNA, it would be trans­lated into ef­fi­cient ex­pres­sion of the ge­nome pro­tein. as­sem­bly and pack­ag­ing of the ge­nome The RNA or DNA com­ple­ment of the (+) strand is called the (−) reg­u ­la­tion and tim­ing of the re­pro­duc­tion cy­cle strand. The (−) strand can­not be trans­lat­ed; it must first be cop­ied mod­u ­la­tion of host de­fenses to make the (+) strand. Ambisense RNA con­tains both (+) and (−) spread to other cells and hosts se­quences. Information not con­tained in vi­ral ge­nomes: A color key for nu­cleic ac­ids, pro­teins, mem­branes, cells, and more is lo­cated in the front of this book. genes en­cod­ing a com­plete pro­tein syn­the­sis ma­chine (e.g., no ri­ bo­somal RNA and no ri­bo­somal or trans­la­tion pro­teins) genes en­cod­ing pro­teins of mem­brane bio­syn­t he­sis telo­meres (to main­tain ge­nomes) or cen­tro­meres (to en­sure seg­re­ga­tion of ge­nomes) this list be­comes shorter with each new edi­tion of this text­ book! DNA Genomes The strat­egy of hav­ing DNA as a vi­ral ge­nome ap­pears at first glance to be the ul­ti­mate in ge­netic ef ­fi­cien­cy: the host ge­netic sys­tem is based on DNA, so vi­ral ge­nome rep­li­ca­tion and ex­ + DNA II pres­sion could sim­ply em­u­late the host sys­tem. While the rep­ li­ca­tion of vi­ral and cel­lu­lar DNA ge­nomes is fun­da­men­tally VI sim­i­lar, the mech­a­nis­tic de­tails are var­ied be­cause vi­ral ge­ ± DNA nomes are struc­tur­a lly di­verse. VII + RNA – DNA ± DNA I Double-Stranded DNA (dsDNA) (Fig. 3.2) There are 38 fam­i­lies of vi­ruses with dsDNA ge­nomes. Those that in­clude ver­te­brate vi­r uses are the Adenoviridae, Alloherpesviridae, Asfarviridae, Herpesviridae, Papillomaviri- + RNA – RNA + mRNA ± RNA dae, Polyomaviridae, Iridoviridae, and Poxviridae. These ge­ IV III nomes may be lin­ear or cir­cu­lar. Genome rep­li­ca­tion and mRNA syn­the­sis are ac­com­plished by host or vi­ral DNA- dependent DNA and RNA po­ly­mer­ases. – RNA V Gapped DNA (Fig. 3.3) Figure 3.1 The Bal­ti­more clas­si­fi­ca­tion. All vi­ruses must pro­duce Members of two vi­rus fam­i­lies, Caulimoviridae and He- mRNA that can be trans­lated by cel­lu­lar ri­bo­somes. This clas­si­fi­ca­tion padnaviridae, have a gapped DNA ge­nome. The Hepadnaviri- sys­tem traces the path­ways from vi­ral ge­nomes to mRNA for the seven clas­ses of vi­ral ge­nomes. dae in­clude vi­r uses that in­fect ver­te­brates. As the gapped DNA ge­nome is par­tially dou­ble stranded, the gaps must be filled to pro­duce per­fect du­plexes. This re­pair pro­cess must pre­cede mRNA syn­the­sis be­cause the host RNA po­ly­mer­ase The seven strat­e­g ies for ex­pres­sion and rep­l i­c a­t ion of vi­ can tran­scribe only fully dsDNA. The un­usual gapped DNA ral ge­nomes are il­lus­t rated in Fig. 3.2 to 3.8. In some cases, ge­nome is pro­duced from an RNA tem­plate by a vi­rus-encoded ge­nomes can en­ter the rep­l i­c a­t ion cy­cle di­rectly, but in oth­ en­zyme, re­verse tran­scrip­tase. ers, ge­nomes must first be re­paired, and vi­ral gene prod­ ucts that par­tic­i­pate in the rep­li­c a­tion cy­cle must first be Single-Stranded DNA (ssDNA) (Fig. 3.4) syn­t he­sized. Examples of spe­cific vi­r uses in each class are Thirteen fam­i­lies of vi­ruses con­tain­ing ssDNA ge­nomes pro­v id­ed. have been rec­og­nized; the fam­i­lies Anelloviridae, Circoviri-  Genomes and Genetics 65 A dsDNA genome ± DNA ± DNA B Polyomaviridae (5 kbp) Ori C Adenoviridae (30–50 kbp) Ori Ori ITR 3' 5' 5' 3' TP ITR D Herpesviridae (120–240 kbp) E Poxviridae (130–375 kbp) L S ITR ITR TRL UL IRL IRS US TRS Terminal loop OriL OriS OriS Figure 3.2 Structure and ex­pres­sion of vi­ral dou­ble-stranded DNA ge­nomes. (A) Synthesis of ge­nomes, mRNA (shown as green line in yel­low box), and pro­tein (shown as brown line). The icon rep­re­sents a poly­oma­vi­rus par­ti­cle. (B to E) Genome con­fig­u­ra­tions. Ori, or­i­gin of rep­li­ca­tion; ITR, in­verted ter­mi­nal re­peat; TP, ter­mi­nal pro­tein; L, long re­gion; S, short re­gion; UL and US, long and short unique re­gions; IRL, in­ter­nal re­peat se­quence, long re­gion; IRS, in­ter­nal re­peat se­quence, short re­gion; TRL, ter­mi­nal re­peat se­quence, long re­gion; TRS, ter­mi­nal re­peat se­quence, short re­gion; OriL, or­i­gin of rep­li­ca­tion of the long re­gion; OriS, or­i­gin of rep­li­ca­tion of the short re­gion. A Gapped, circular, dsDNA genome B Hepadnaviridae (3–3.3 kbp) (+) 3' (–) ± DNA ± DNA 5' 5' 3' + RNA – DNA ± DNA Figure 3.3 Structure and ex­pres­sion of vi­ral gapped, cir­cu­lar, dou­ble-stranded DNA ge­nomes. (A) Synthesis of ge­nome, mRNA, and pro­tein. (B) Configuration of the hepadnavirus ge­nome. dae, Genomoviridae, and Parvoviridae in­clude vi­ruses that tem­plates (Box 3.3). One so­lu­tion to this prob­lem is that RNA in­fect ver­te­brates. ssDNA must be cop­ied into mRNA be­fore vi­rus ge­nomes en­code RNA-dependent RNA po­ly­mer­ases that pro­teins can be pro­duced. However, RNA can be made only pro­duce RNA from RNA tem­plates. The other so­lu­tion, ex­em­ from a dsDNA tem­plate, what­ever the sense of the ssDNA. pli­fied by ret­ro­vi­rus ge­nomes, is re­verse tran­scrip­tion of the Consequently, DNA syn­t he­sis must pre­cede mRNA pro­duc­ ge­nome to dsDNA, which can be tran­scribed by host RNA tion in the rep­li­ca­tion cy­cles of these vi­ruses. All syn­t he­sis of po­ly­mer­ase. vi­ral DNA is cat­a ­lyzed by cel­lu­lar DNA po­ly­mer­ases. dsRNA (Fig. 3.5) RNA Genomes There are twelve fam­i­lies of vi­r uses with lin­ear dsRNA Cells have no RNA-dependent RNA po­ly­mer­ases that can rep­ ge­nomes. The num­ber of dsRNA seg­ments in the vi­rus par­ li­cate the ge­nomes of RNA vi­ruses or make mRNA from RNA ti­cle may be 1 (Totiviridae, Hypoviridae, and Endornaviridae, 66 Chapter 3 A ssDNA genome or + DNA – DNA + DNA ± DNA – DNA B Circoviridae (1.8–2.3 kb) C Parvoviridae (4–6 kb) B B' A A' A' D D' A C C' Figure 3.4 Structure and ex­pres­sion of vi­ral sin­gle-stranded DNA ge­nomes. (A) Synthesis of ge­nomes, mRNA, and pro­tein. (B and C) Genome con­fig­u­ra­tions. A dsRNA genome B Reoviridae (18.2–30.5 kbp in 1–12 dsRNA segments) L1 L2 L3 3' 5' 3' 5' 3' 5' 5' c 3' 5' c 3' 5' c 3' RNA M1 M2 M3 3' 5' 3' 5' 3' 5' 5' c 3' 5' c 3' 5' c 3' RNA S1 S2 S3 S4 3' 5' 3' 5' 3' 5' 3' 5' 5' c 3' 5' c 3' 5' c 3' 5' c 3' Figure 3.5 Structure and ex­pres­sion of vi­ral dou­ble-stranded RNA ge­nomes. (A) Synthesis of ge­nomes, mRNA, and pro­tein. (B) Genome con­fig­ur­ a­tion. B OX 3.3 B A C K G R O U N D RNA syn­the­sis in cells There are no known host cell en­z ymes that can copy the ge­nomes of RNA vi­r uses. How­ ever, at least one en­z yme, RNA po­ly­mer­ase II, can copy an RNA tem­plate. The 1.7-kb cir­ cu­lar, ssRNA ge­nome of hep­a­ti­tis delta sat­el­ (–) strand lite vi­r us is cop­ied by RNA po­ly­mer­ase II to genome RNA form multimeric RNAs (see the fig­u re). How RNA po­ ly­ mer­ase II, an en­ z yme that pro­ duces pre-mRNAs from DNA tem­plates, is re­pro­grammed to copy a cir­cu­lar RNA tem­ plate is not known. Hepatitis delta sat­el­lite (−) strand ge­nome RNA is cop­ied by RNA po­ly­mer­ase II at the in­di­cated po­si­tion. The po­ly­mer­a se passes the po­ly(A) sig­nal (pur­ple box) and the self-cleavage do­main (red cir­cle). For more in­for­ma­t ion, see Fig. 6.25. Redrawn from Tay­ lor JM. 1999. Curr Top Microbiol Immunol 239:107–122, with per­mis­sion.  Genomes and Genetics 67 A ss (+) RNA brates, plants, and ver­te­brates). While dsRNA con­tains a (+) strand, it can­not be trans­lated to syn­t he­size vi­ral pro­teins as part of a du­plex. The (−) strand of the ge­no­mic dsRNA is first cop­ied into mRNAs by a vi­ral RNA-dependent RNA po­ly­ Genome mer­ase. Newly syn­the­sized mRNAs are encapsidated and then cop­ied to pro­duce dsRNAs. – RNA (+) Strand RNA (Fig. 3.6) There are more dif­fer­ent types of (+) strand RNA vi­ruses B Coronaviridae (27.6–41.1 kb) than any other, and 38 fam­i­lies have been rec­og­nized [not count­ing (+) strand RNA vi­ruses with DNA in­ter­me­di­ates]. 5’ c AnAOH3’ These ge­nomes are lin­ear and may be sin­gle mol­e­cules (non­ UTR UTR segmented) or seg­mented, de­pend­ing on the fam­ily. The fam­ i­lies Arteriviridae, Astroviridae, Caliciviridae, Coronaviridae, Flaviviridae (9.6–12.3 kb) Flaviviridae, Hepeviridae, Nodaviridae, Picornaviridae, and 5’ c 3’ Togaviridae in­clude vi­ruses that in­fect ver­te­brates. (+) strand UTR UTR RNA ge­nomes usu­a lly can be trans­lated di­rectly into pro­tein Picornaviridae (6.7–9.07 kb) by host ri­bo­somes. The ge­nome is rep­li­cated in two steps. The (+) strand ge­nome is first cop­ied into a full-length (−) strand, 5’ cVPg AnAOH3’ and the (−) strand is then cop­ied into full-length (+) strand UTR UTR ge­nomes. In some cases, a subgenomic mRNA is pro­duced. Togaviridae (9.7–11.8 kb) (+) Strand RNA with a DNA Intermediate (Fig. 3.7) 5’ c AnAOH3’ Members of four vi­rus fam­i­lies are (+) strand RNA vi­ruses UTR UTR with a DNA in­ter­me­di­ate; those vi­ruses within Retroviridae Figure 3.6 Structure and ex­pres­sion of vi­ral sin­gle-stranded in­fect ver­te­brates. In con­trast to other (+) strand RNA vi­ruses, (+) RNA ge­nomes. (A) Synthesis of ge­nomes, mRNA, and pro­tein. (B) the (+) strand RNA ge­nome of ret­ro­vi­ruses is con­verted to a Genome con­fig­u­ra­tions. UTR, un­trans­lated re­gion; VPg, vi­rion pro­tein, ge­nome linked. dsDNA in­ter­me­di­ate by vi­ral RNA-dependent DNA po­ly­mer­ ase (re­verse tran­scrip­tase). Following in­te­gra­tion into host DNA, the vi­ral DNA then serves as the tem­plate for vi­ral vi­ruses of fungi, pro­to­zoa, and plants); 2 (Partitiviridae, mRNA and ge­nome RNA syn­the­sis by cel­lu­lar en­zymes. Birnaviridae, and Megabirnaviridae, vi­ r uses of fungi, plants, in­sects, fish, and chick­ens); 3 (Cystoviridae, vi­ruses of (−) Strand RNA (Fig. 3.8) Pseudomonas bac­te­ria); 4 (Chrysoviridae, vi­ruses of fun­gi); Viruses with (−) strand RNA ge­nomes are found in 19 or 10 to 12 (Reoviridae, vi­r uses of pro­to­zoa, fungi, in­ver­te­ fam­i­lies. These ge­nomes are lin­ear and may be sin­gle mol­e­cules A ss (+) RNA with DNA intermediate + RNA – DNA DNA + RNA B Retroviridae (7–11 kb) U5 U3 5’ c AnAOH3’ Figure 3.7 Structure and ex­pres­sion of vi­ral sin­gle-stranded (+) RNA ge­nomes with a DNA in­ter­me­di­ate. (A) Synthesis of ge­nomes, mRNA, and pro­tein. (B) Genome con­fig­u­ra­tion. 68 Chapter 3 A ss (–) RNA – RNA + RNA – RNA B Segmented genomes: Orthomyxoviridae C Ambisense (–) strand RNA (10–15 kb in 6–8 RNAs) Arenaviridae (11 kb in 2 RNAs) (–) strand RNA segments Peribunyaviridae (12.4–16.6 kb in 3 RNAs) 1 2 3 4 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ L RNA 5’ c 3’ 5 6 7 8 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Nonsegmented genomes: Paramyxoviridae (15.1–18.2 kb) M RNA 5’ c 3’ 3’ 5’ Rhabdoviridae (11–15 kb) S RNA 5’ c 3’ 3’ 5’ Figure 3.8 Structure and ex­pres­sion of vi­ral sin­gle-stranded (−) RNA ge­nomes. (A) Synthesis of ge­nomes, mRNA, and pro­ tein. The icon rep­re­sents an or­t ho­myxo­v i­rus par­ti­cle. (B and C) Genome con­fig­u­ra­tions. (nonsegmented; some vi­ruses with this con­fig­u­ra­tion have pro­teins or en­zymes. A fun­da­men­tal dif­fer­ence be­tween the been clas­si­fied in the or­der Mononegavirales) or seg­mented. ge­nomes of vi­ruses and those of their hosts is that al­t hough Viruses of this type that can in­fect ver­te­brates in­clude mem­ vi­ral ge­nomes are of­ten cov­ered with pro­teins, they are usu­ bers of the Arenaviridae, Bornaviridae, Filoviridae, Hanta- ally not bound by his­tones in the vi­rus par­ti­cle (polyomaviral viridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and papillomaviral ge­nomes are ex­cep­tions). However, it is and Rhabdoviridae fam­ i­lies. Unlike (+) strand RNA, (−) likely that all­vi­ral DNAs be­come coated with his­tones shortly strand RNA ge­nomes can­not be trans­lated di­rectly into pro­ af­ter they en­ter the nu­cle­us. tein but must be first cop­ied to make (+) strand mRNA. While vi­ral ge­nomes are all­nu­cleic ac­ids, they should not There are no en­zymes in the cell that can make mRNAs from be thought of as one-dimensional struc­tures. Virology text­ the RNA ge­nomes of (−) strand RNA vi­ruses. These vi­rus par­ books (this one in­cluded) of­ten draw ge­nomes as straight, ti­cles there­fore con­tain vi­rus-encoded RNA-dependent RNA one-dimensional lines, but this no­ta­tion is for il­lus­tra­tive pur­ po­ly­mer­ases. The ge­nome is also the tem­plate for the syn­t he­ poses on­ly; phys­i­cal re­a l­ity is cer­tain to be dra­mat­i­cally dif­ sis of full-length (+) strands, which, in turn, are cop­ied to pro­ fer­ent. Genomes have the po­ ten­tial to adopt amaz­ ing duce (−) strand ge­nomes. sec­ond­ary and ter ­tiary struc­tures in which nu­cle­o­tides may The ge­nomes of cer­tain (−) strand RNA vi­ruses (e.g., mem­ en­gage in long-distance in­ter­ac­tions (Fig. 3.9). bers of the Arenaviridae and Bunyaviridae) are ambisense: The se­quences and struc­tures near the ends of vi­ral ge­ they con­tain both (+) and (−) strand in­for­ma­tion on a sin­gle nomes are of­ten in­dis­pens­able for rep­li­ca­tion. For ex­am­ple, the strand of RNA (Fig. 3.8C). The (+) sense in­for­ma­tion in the DNA se­quences at the ends of par­vo­vi­rus ge­nomes form T- ge­nome is trans­lated upon en­try of the vi­ral RNA into cells. shaped struc­tures that are re­quired for prim­ing dur­ing DNA Replication of the RNA ge­nome yields ad­di­tional (+) sense se­ syn­the­sis. Proteins co­va­lently at­tached to 5′ ends, in­verted and quences, which are then trans­lat­ed. tan­dem re­peats, and bound tRNAs may also par­tic­i­pate in the rep­ li­ ca­ tion of RNA and DNA ge­ nomes. Secondary RNA What Do Viral Genomes Look Like? struc­tures may fa­cil­i­tate trans­la­tion (the in­ter­nal ri­bo­some Some small RNA and DNA ge­nomes en­ter cells from vi­r us en­try site [IRES] of pi­cor­na­v i­rus ge­nomes) and ge­nome pack­ par­ti­cles as na­ked mol­e­cules of nu­cleic acid, whereas oth­ers ag­ing (the struc­tured pack­ag­ing sig­nal of ret­ro­v i­ral ge­nomes, are al­ways as­so­ci­ated with spe­cial­ized nu­cleic ac­id-binding [Fig. 3.9]).  Genomes and Genetics 69 A Linear (+) strand RNA genome of a picornavirus 5’ VPg AnAOH3’ UTR UTR B 5’ 3’ 4252 C D SL1 SL4 SL2 SL3 TAR pA U5 PBS DIS SD Ψ AUG Figure 3.9 Genome struc­tures in car­toons and in real life. (A) Linear rep­re­sen­ta­t ion of a pi­cor­na­v i­r us RNA ge­nome. UTR, un­ trans­lated re­gion. (B) Long-distance RNA-RNA in­ter­ac­tions in a tombusvirus RNA ge­nome. The 4,252-nucleotide vi­ral ge­nome is shown with sec­ond­ary RNA struc­tures at the 5′ and 3′ ends. Sequences that base-pair are shown in blue (re­quired for RNA frameshifting) and red (re­quired to bring ri­bo­somes from the 3′ end to the 5′ end). Courtesy of Anne Si­mon, University of Mary­land. (C) Schematic rep­re­sen­ ta­tion of RNA sec­ond­a ry-structure el­e­ments in the hu­man im­mu­no­de­fi­ciency vi­r us type 1 5′ leader, in­clud­i ng the core pack­ag­i ng sig­nal. (D) NMR struc­t ure of the RNA shown in C, with­out­el­e­ments col­ored black. Courtesy of Paul Bieniasz, Rocke­fel­ler University. Coding Strategies syn­t he­sis, leaky scan­ning, sup­pres­sion of ter­mi­na­tion, and The com­pact ge­nome of most vi­ruses ren­ders the “one gene, ri­bo­somal frameshifting. In gen­eral, the smaller the ge­nome, one mRNA” dogma in­ac­cu­rate. Extraordinary tac­tics for in­ the greater the com­pres­sion of ge­netic in­for­ma­tion. for­ma­tion re­trieval, such as the pro­duc­tion of mul­ti­ple subge­ nomic mRNAs, al­ter­na­tive mRNA splic­ing, RNA ed­it­ing, and What Can Viral Sequences Tell Us? nested tran­scrip­tion units (Fig. 3.10), al­low the pro­duc­tion of Knowledge about the phys­i­cal na­ture of ge­nomes and cod­ing mul­ti­ple pro­teins from a sin­g le vi­ral ge­nome. Further ex­ strat­e­gies was first ob­tained by the study of the nu­cleic ac­ids pan­sion of the cod­ing ca­pac­ity of the vi­ral ge­nome is achieved of vi­ruses. Indeed, DNA se­quenc­ing tech­nol­ogy was per­ by post­t ran­scrip­t ional mech­a­n isms, such as polyprotein fected on vi­ral ge­nomes. The first ge­nome of any kind to be  Figures in Mechanism Diagram Virus Chapter(s) appendix Multiple 3' 5' Genome Adenoviridae 7, 8 1, 2 subgenomic Hepadnaviridae 7, 10 11, 12 mRNAs Herpesviridae 7 5' c 5' c 5' c 5' c 5' c mRNAs Paramyxoviridae 6 17, 18 Poxviridae 7 25, 26 Proteins Rhabdoviridae 6 31, 32 Alternative Adenoviridae 7, 8 1, 2 5' c mRNA splicing Orthomyxoviridae 8 15, 16 5' c Papillomaviridae 7, 8 5' c Polyomaviridae 7, 8 23, 24 Retroviridae 8, 10 29, 30 RNA editing Editing site Paramyxoviridae 6, 8 Viral genome Filoviridae 8 5' c 3' mRNA 1 Hepatitis delta 8 Protein 1 virus 5' c 3' mRNA 2 (+1 G) Protein 2 Information on CBF USF +1 3' Adenoviridae 7–9 1, 2 both strands Polyomaviridae 7–9 23, 24 Retroviridae 10 29, 30 Double-stranded DNA Proteins Polyprotein Viral gene Alphaviruses 6, 11 33, 34 synthesis mRNA Flaviviridae 6, 11 9, 10 Picornaviridae 6, 11 21, 22 Polyprotein Retroviridae 6, 11 29, 30 Processing Leaky scanning Viral gene Orthomyxoviridae 11 15, 16 AUG AUG Paramyxoviridae 11 mRNA Polyomaviridae 11 Retroviridae 11 29, 30 Proteins Reinitiation Viral gene Orthomyxoviridae 11 15, 16 Herpesviridae 11 mRNA Proteins Suppression of Viral gene Alphaviruses 11 33, 34 termination Stops Retroviridae 11 29, 30 mRNA Proteins Ribosomal Viral gene Astroviridae 11 frameshifting Frameshift site Coronaviridae 11 5, 6 Retroviridae 11 29, 30 mRNA Upstream of frameshift site Downstream of frameshift site Proteins IRES Viral gene Flaviviridae 11 Picornaviridae 11 21, 22 mRNA Proteins Nested mRNAs 2a S Sa M Coronaviridae 6 5, 6 5' 3' Viral gene Arteriviridae 6 5, 6 HE 4 E N 2a S Sa M 5' c AnAOH3' Protein HE 4 E N S Sa M AnAOH3' Protein 5' c HE 4 E N S Sa M 5' c AnAOH3' Protein 4 E N Figure 3.10 Information re­trieval from vi­ral ge­nomes. Different strat­e­gies for de­cod­ing the in­for­ma­tion in vi­ral ge­nomes are de­picted. CBF, CCAAT-binding fac­tor; USF, up­stream stim­u ­la­tory fac­tor; IRES, in­ter­nal ri­bo­some en­try site.  Genomes and Genetics 71 se­ quenced was that of the Escherichia coli bac­te­rio­phage around us (es­pe­cially in the sea) is as­tro­nom­i­cal. Most are un­ MS2, a lin­ear ssRNA of 3,569 nu­cle­o­tides. dsDNA ge­nomes characterized and, be­cause their hosts are also un­k nown, of larger vi­ruses, such as her­pes­v i­ruses and pox­v i­ruses (vac­ can­not be in­ves­ti­gated. A re­duc­tion­ist study of in­di­vid­ual cinia vi­rus), were se­quenced com­pletely by the 1990s. Since com­po­nents in iso­la­tion pro­vi­des few an­swers. Although the then, high-throughput se­quenc­ing has rev­o­lu­tion­ized the bi­ re­duc­tion­ist ap­proach is of­ten the sim­plest ex­per­i­men­tally, it o­log­i­cal sci­ences, al­low­ing rapid de­ter­mi­na­tion of ge­nome is also im­por­tant to un­der­stand how the ge­nome be­haves se­quences from clin­i­cal and en­v i­ron­men­tal sam­ples. Organ- among oth­ers (pop­u­la­tion bi­­ol­ogy) and how the ge­nome and tis­sue-specific viromes of many or­gan­isms have been changes with time (evo­lu­tion). Nevertheless, re­duc­tion­ism has de­ter­mined. In one study, over 186 host spe­cies rep­re­sent­ing pro­v ided much-needed de­t ailed in­for­ma­t ion for trac­t a­ble the phy­lo­ge­netic di­ver­sity of ver­te­brates, in­clud­ing lance­lets vi­rus-host sys­tems. These sys­tems al­low ge­netic and bio­chem­ (chor­dates, but con­sid­ered in­ver­te­brates), jaw­less fish, car­ti­ i­cal an­a­ly­ses and pro­vide mod­els of in­fec­tion in vi­vo and in lag­i­nous fish, ray-finned fish, am­phib­i­ans, and rep­tiles, all­ cells in cul­ture. Unfortunately, vi­ruses and hosts that are dif ­fi ­ an­ces­tral to birds and mam­mals, were sam­pled. RNA was ex­ cult or im­pos­si­ble to ma­nip­u­late in the lab­o­ra­tory re­main un­ tracted from mul­ti­ple or­gans and sub­jected to high-through­ der­stud­ied or ig­nored. put se­quenc­ing. Among 806 bil­lion ba­ses that were read, 214 new vi­ral ge­nomes were iden­ti­fied. The re­sults show that in The “Big and Small” of Viral ver­te­brates other than birds and mam­mals, RNA vi­ruses are Genomes: Does Size Matter? more nu­mer­ous and di­verse than sus­pected. Every vi­ral fam­ The ques­tion “does ge­nome size mat­ter” is dif ­fi­cult to an­swer ily or ge­nus of bird and mam­mal vi­ruses is also rep­re­sented con­sid­er­ing the three or­ders of mag­ni­tude in ge­nome length in vi­ruses of am­phib­i­ans, rep­tiles, or fish. Arenaviruses, fi­lo­ that sep­a­rate the larg­est and the small­est vi­ral ge­nomes. The vi­ruses, and han­ta­v i­ruses were found for the first time in two larg­est vi­ral ge­nomes known are those of Pandoravirus aquatic ver­te­brates. The ge­nomes of some fish vi­ruses have now salinus (2.4 mil­lion ba­ses of dsDNA) and Pandoravirus dulcis ex­panded so that their phy­lo­ge­netic di­ver­sity is larger than in (1.9 mil­lion ba­ses of dsDNA), en­cod­ing 2,541 and 1,487 open mam­ma­lian vi­ruses. New rel­a­tives of in­flu­enza vi­ruses were read­ing frames, re­spec­tively. The larg­est RNA vi­rus ge­nomes found in hag­fish, am­phib­i­ans, and ray-finned fish. As of this are far be­hind (Box 3.4). At the other end are anelloviruses, writ­ing, the com­plete se­quences of >8,000 dif­fer­ent vi­ral ge­ with a 1,759-base ssDNA ge­nome en­cod­ing two pro­teins (Fig. nomes have been de­ter­m ined. Published vi­ral ge­nome se­ 3.3B), and vi­roids, cir­cu­lar, sin­gle-stranded RNA mol­e­cules of quences can be found at http://​w ww.​ncbi.​nlm.​nih.​gov/​genome​ 246 to 401 nu­cle­o­tides that en­code no pro­tein (Volume II, /­v iruses/​. Chapter 13). Anelloviruses in­clude ag­ri­cul­tur­a lly im­por­tant The util­ity of vi­ral ge­nome se­quences ex­tends well be­yond path­o­gens of chick­ens and pigs and torque teno (TT) vi­rus, build­ing a cat­a ­log of vi­ruses. These se­quences are the pri­ which in­fects >90% of hu­mans with no known con­se­quence. mary ba­sis for clas­si­fi­ca­tion and also pro­v ide in­for­ma­tion on Viroids cause eco­nom­i­cally im­por­tant dis­eases of crop plants. the or­i­gin and evo­lu­tion of vi­ruses. In out­­breaks or ep­i­dem­ All vi­ruses with ge­nome sizes span­ning the range from the ics of vi­ral dis­ease, even par­tial ge­nome se­quences can pro­ big­gest to the small­est are suc­cess­ful as they con­tinue to re­pro­ vide in­for­ma­tion about the iden­tity of the in­fect­ing vi­rus and duce and spread within their hosts. Despite de­tailed an­a­ly­ses, its spread in dif­fer­ent pop­u ­la­tions. New vi­ral nu­cleic acid se­ there is no ev­i­dence that one size is more ad­van­ta­geous than quences can be as­so­ci­ated with dis­ease and char­ac­ter­ized an­other. All vi­ral ge­nomes have evolved un­der re­lent­less se­lec­ even in the ab­sence of stan­dard vi­ro­log­i­cal tech­niques (Vol­ tion, so ex­tremes of size must pro­vide par­tic­u­lar ad­van­tages. ume II, Chapter 10). For ex­am­ple, hu­man her­pes­v i­rus 8 was One fea­ture dis­tin­guish­ing large ge­nomes from smaller ones is iden­ti­fied by com­par­ing se­quences pres­ent in dis­eased and the pres­ence of many genes that en­code pro­teins for vi­ral ge­ nondiseased tis­sues, and a novel mem­ber of the par­vo­v i­rus nome rep­li­ca­tion, nu­cleic acid me­tab­o­lism, and coun­ter­ing fam­ily was iden­ti­fied as the cause of un­ex­pected deaths of host de­fense sys­tems. When mimiviruses were first dis­cov­ lab­o­ra­tory mice in Aus­tra­lia and the United States. ered, the sur­prise was that their ge­nomes en­coded com­po­nents Despite their util­ity, ge­nome se­quences can­not pro­vide a of the pro­tein syn­the­sis sys­tem, such as tRNAs and aminoacyl- com­plete un­der­stand­ing of how vi­ruses re­pro­duce. The ge­ tRNA syn­the­tases. Tupanviruses, iso­lated from soda lakes in nome se­quence of a vi­rus is at best a bi­o­log­i­cal “parts list”: it Bra­zil and deep ocean sed­i­ments, en­code all­ 20 aminoacyl- pro­vi­des some in­for­ma­tion about the in­trin­sic prop­er­ties of a tRNA syn­the­tases, 70 tRNAs, mul­ti­ple trans­la­tion pro­teins, vi­rus (for ex­am­ple, pre­dicted se­quences of vi­ral pro­teins and and more. Only the ri­bo­some is lack­ing. Why would large vi­ par­ti­cle com­po­si­tion), but says lit­tle or noth­ing about how the ral ge­nomes carry these genes when they are avail­­able in their vi­rus in­ter­acts with cells, hosts, and pop­u­la­tions. This lim­i­ta­ cel­lu­lar hosts? Perhaps by pro­duc­ing a large part of the trans­ tion is best il­lus­trated by the re­sults of en­vi­ron­men­tal metage­ tional ma­ la­ chin­ ery, vi­ ral mRNAs can be more ef ­fi­ ciently nomic an­a­ly­ses, which re­veal that the num­ber of vi­ruses trans­lated. This ex­pla­na­tion is con­sis­tent with the find­ing that 72 Chapter 3 B OX 3.4 E X P E R I M E N T S Planaria and mol­lusks yield the big­gest RNA ge­nomes In the past 20 years the de­vel­op­ment of high- find larger vi­rus RNAs, sug­gest­ing that we an RNA ge­nome of 35,906 nu­cle­o­tides with throughput nu­cleic acid se­quenc­ing meth­ods have not yet reached the size limit of RNA ge­ ORFs that en­code two polyproteins. has rap­idly in­creased the pace of vi­rus dis­cov­ nomes. From the per­spec­tive of ge­nome size, the ery. Yet in that time, while the larg­est DNA A close study of the transcriptome of a dis­cov­ery of these nidovirus ge­nomes sug­gests ge­nomes have in­creased nearly ten times, the pla­nar­ian re­vealed a new nidovirus, pla­nar­ that vi­ruses with even larger RNAs re­main to larg­est known RNA vi­ral ge­nome has only in­ ian se­cre­tory cell nidovirus, with an RNA ge­ be dis­cov­ered. In both cases the vi­ruses were creased in size by ten per­cent. This sit­u­a­tion nome of 41,103 nu­cle­o­t ides. This vi­ral ge­nome iden­ti­fied from se­quences that had been de­ has now changed with the dis­cov­ery of new is un­usual be­cause it en­codes a sin­gle, long pos­ited in pub­lic da­ta­bases, al­though in both RNA vi­ruses of pla­nar­i­a ns and mol­lusks. open read­ing frame of 13,556 amino ac­ids— cases, in­ fec­ tious vi­ ruses were not re­ ported. Until very re­cently, the big­gest RNA vi­rus the lon­gest vi­ral open read­ing frame (ORF) Nevertheless, many or­ gan­isms have not yet ge­nome known was 33.5 kb (ball py­thon ni­ dis­cov­ered so far. All the other known nido­ had their ge­nomes se­quenced and it is likely dovirus), which is much larger than the av­er­ viruses en­code mul­t i­ple open read­ing frames. that many RNA vi­ruses re­main to be dis­cov­ age sized RNA vi­rus ge­nome of 10 kb. The Phylogenetic anal­y­sis of known nidoviruses ered. Declaring an up­per limit on RNA ge­ rea­son for the dif­fer­ence is that RNA po­ly­ sug­gests that the pla­nar­ian vi­rus arose from nome size does not seem rea­son­able if we have mer­ases make er­rors, and most do not have vi­ruses with mul­t i­ple ORFs, af­ter which their not sam­pled ev­ery spe­cies. proof­read­ing ca­pa­bil­i­ties. Nidovirus ge­nomes sin­gle ORF ex­panded in size. Saberi A, Gulyaeva AA, Brubacher JL, Newmark PA, en­code a proof­read­ing exoribonuclease which The other nidovirus with a large RNA ge­ Gorbalenya AE. 2018. A pla­ nar­ ian nidovirus ex­ im­proves rep­li­ca­tion fi­del­ity and pre­sum­ably nome was dis­ cov­ ered by search­ ing all­the pands the lim­its of RNA ge­nome size. PLoS Pathog al­lows for larger ge­nomes. Even with a proof­ avail­­able RNA se­quences of the mol­lusk Aply- 14:e1007314. read­ing en­z yme, the big­gest RNA vi­rus ge­ sia californica. With a sim­ple ner­vous sys­tem Debat HJ. 2018. Expanding the size limit of RNA vi­ nome is much smaller than the min­ mal i­ of 20,000 neu­ rons, this mol­ lusk has been rus­es: ev­i­dence of a novel di­ver­gent nidovirus in Cal­i­ for­nia sea hare, with a ∼39.5 kb vi­rus ge­nome. bioRxiv cel­lu­lar DNA ge­nome, which is 200 kb. The stud­ied as a model sys­tem in many lab­o­ra­to­ 307678. re­sults of two new stud­ies show that we can ries. Aplysia californica nido-like vi­rus has the co­don and amino acid us­age of tupanvirus is dif­fer­ent trans­la­tion ma­chin­ery, as well as host cell sys­tems to make from that of the amoeba that it in­fects. mem­branes and gen­er­ate en­er­g y. Another in­trigu­ing set of genes be­longs to tetraselmis The pa­ram­e­ters that limit the size of vi­ral ge­nomes are ­v i­rus 1, which in­fects green al­gae. These hosts, found in nu­tri­ largely un­known. There are cel­lu­lar DNA and RNA mol­e­cules ent-rich ma­rine and fresh wa­ters, are pho­to­syn­thetic. The that are much lon­ger than those found in vi­rus par­ti­cles. Con­ vi­ral ge­nome en­codes py­ru­vate for­ma­te-lyase and py­ru­vate sequently, the rate of nu­cleic acid syn­the­sis is not likely to be for­ma­te-lyase-activating en­zyme, which are key mem­bers of lim­it­ing. Nor does the cap­sid vol­ume ap­pear to limit ge­nome size: cel­lu­lar an­aer­o­bic res­pi­ra­tion path­ways and al­low en­ergy pro­ the ico­sa­he­dral shell of Mimivirus, which houses a 1.2 mil­lion- duc­tion when no ox­y­gen is avail­­able. Green al­gae may use this base-pair DNA ge­nome, is con­structed mainly of a sin­gle ma­jor sys­tem in wa­ters de­pleted of ox­y­gen by ex­u­ber­ant al­gal cap­sid pro­tein. For larger ge­nomes, the so­lu­tion is he­li­cal sym­ growth. If this pro­cess oc­curs in cells, why does the vi­ral ge­ me­try, which can in prin­ci­ple ac­com­mo­date very large ge­ nome carry some of the genes in­volved? The an­swer is not nomes. The Pandoraviruses, with the larg­est known DNA vi­ral known, but it is pos­si­ble that the ex­tra met­a­bolic de­mands ge­nomes (2,500 kbp), are housed in de­cid­edly nonisometric placed on cells dur­ing vi­rus rep­li­ca­tion—es­pe­cially at night— ovoid par­ti­cles 1 μm in length and 0.5 μm in di­am­e­ter. re­quire ad­di­tional fer­men­ta­tion en­zymes for en­ergy pro­duc­ There is no rea­son to be­lieve that the up­per limit in vi­ral tion. The pres­ence of these genes sug­gests that tetraselmis par­ti­cle and ge­nome size has been dis­cov­ered. The core com­ vi­r us 1 can change host me­tab­o­l ism, per­haps fa­cil­i­tat­i ng its part­ment of a mimivirus par­ti­cle is larger than needed to ac­ re­pro­duc­tion. com­mo­date the 1,200-kbp DNA ge­nome. A par­ti­cle of this size These large vi­ruses there­fore have suf ­fi­cient cod­ing ca­pac­ could, in prin­ci­ple, house a ge­nome of 6 mil­lion bp if the DNA ity to es­cape some re­stric­tions im­posed by host cell bio­chem­ were packed at the same den­sity as in poly­oma­vi­ruses. Indeed, is­try. The small­est ge­nome of a free-liv­ing cell is pre­dicted to if the ge­nome were packed into the par­ti­cle at the den­sity com­prise 12 mil­lion bp, the Remarkably, this num­ber is smaller than the ge­netic con­tent size of that of the small­est free-liv­ing uni­cel­lu­lar eu­kary­ote. of large vi­ral DNA ge­nomes. Nevertheless, the big vi­ruses are In cells, DNAs are much lon­ger than RNA mol­e­cules. RNA not cells: their re­pro­duc­tion ab­so­lutely re­quires the cel­lu­lar is less sta­ble than DNA, but in the cell, much of the RNA is used  Genomes and Genetics 73 for the syn­the­sis of pro­teins and there­fore need not ex­ceed the isms might have had RNA ge­nomes. Viruses with RNA ge­ size needed to spec­ify the larg­est po­ly­pep­tide. However, this nomes might have evolved dur­ ing this time. Later, DNA con­straint does not ap­ply to vi­ral ge­nomes. Yet the larg­est vi­ral re­placed RNA as cel­lu­lar ge­nomes, per­haps through the ac­tion sin­gle-molecule RNA ge­nomes, the 41-kb (+) strand RNAs of of re­verse tran­scrip­tases. With the emer­gence of DNA ge­ the nidoviruses (Box 3.4), are dwarfed by the larg­est (2,500- nomes prob­a­bly came the evo­lu­tion of DNA vi­ruses. However, kbp) DNA vi­rus ge­nomes. Susceptibility of RNA to chem­i­cal those with RNA ge­nomes were and re­main evo­lu­tion­arily and nu­cle­ase at­tack might limit the size of vi­ral RNA ge­nomes. com­pet­i­tive, and hence they con­tinue to sur­vive to this day. However, the most likely ex­pla­na­tion is that there are few Analysis of se­quences of more than 4,000 RNA-dependent known en­zymes that can cor­rect er­rors in­tro­duced dur­ing RNA po­ly­mer­ases is con­sis­tent with the hy­poth­e­sis that the RNA syn­the­sis. An exo­nu­cle­ase en­coded in the co­ro­na­vi­rus ge­ first RNA vi­ruses to emerge af­ter the evo­lu­tion of trans­la­tion nome is one ex­cep­tion: its pres­ence could ex­plain the large size were those with (+) strand RNA ge­nomes. The last com­mon of these RNAs. DNA po­ly­mer­ases can elim­i­nate er­rors dur­ing an­ces­tor of these vi­ruses en­coded only an RNA-dependent po­ly­mer­i­za­tion, a pro­cess known as proof­read­ing, and re­main­ RNA po­ly­mer­ase and a sin­gle cap­sid pro­tein. Double-stranded ing er­rors can also be cor­rected af­ter DNA syn­the­sis is com­ RNA vi­ruses evolved from (+) strand RNA vi­ruses on at least plete. The av­er­age er­ror fre­quen­cies for RNA ge­nomes are about two dif­fer­ent oc­ca­sions, and (−) strand RNA vi­ruses evolved 1 misincorporation in 104 or 105 nu­cle­o­tides po­ly­mer­ized. In an from dsRNA vi­ruses. The emer­gence of vi­ruses with the lat­ter RNA vi­ral ge­nome of 10 kb, a mu­ta­tion fre­quency of 1 in 104 ge­nome types was likely fa­cil­i­tated by the cap­ture of genes such would pro­duce about 1 mu­ta­tion in ev­ery rep­li­cated ge­nome. as those en­cod­ing RNA helicases, to al­low for the pro­duc­tion Hence, very long vi­ral RNA ge­nomes, per­haps lon­ger than 40 of larger ge­nomes. kb, would sus­tain too many mu­ta­tions that would be le­thal. Single-stranded DNA vi­ruses of eu­kary­otes ap­pear to have Even the 7.5-kb ge­nome of po­lio­vi­rus ex­ists at the edge of in­fec­ evolved from genes con­trib­uted from both bac­te­rial plas­mids tiv­i­ty: treat­ment of the vi­rus with the RNA mu­ta­gen ri­ba­vi­rin and (+) strand RNA vi­ruses. Different dsDNA vi­ruses orig­i­ causes a >99% loss in a sin­gle round of rep­li­ca­tion. nated from bac­te­rio­phages at least twice. The larger eu­kary­otic When new vi­ral ge­nomes are dis­cov­ered, of­ten many of the DNA vi­ruses form a mono­phy­letic group based on anal­y­sis of pu­ta­tive genes are pre­vi­ously un­k nown. For ex­am­ple, >93% of 40 genes that de­rive from a last com­mon an­ces­tor. These vi­ the >2,500 genes of Pandoravirus salinus re­sem­ble noth­ing ruses ap­pear to have emerged from smaller DNA vi­ruses by known, and 453 of the 663 pre­dicted open read­ing frames of the cap­ture of mul­ti­ple eu­kary­otic and bac­te­rial genes, such as tetraselmis vi­rus 1 show no se­quence sim­i­lar­ity to known those en­cod­ing trans­la­tion sys­tem com­po­nents. pro­teins. The im­pli­ca­tion of these find­ings is clear: our ex­plo­ There is no ev­i­dence that vi­ruses are mono­phy­letic, i.e., de­ ra­tion of global ge­nome se­quences is far from com­plete, and scended from a com­mon an­ces­tor: there is no sin­gle gene vi­ruses with larger ge­nomes might yet be dis­cov­ered. shared by all­vi­ruses. Nevertheless, vi­ruses with dif­fer­ent ge­ nomes and rep­li­ca­tion strat­e­gies do share a small set of vi­ral The Origin of Viral Genomes hall­mark genes that en­code ico­sa­he­dral cap­sid pro­teins, nu­ The ab­sence of bona fi­de vi­ral fos­sils, i.e., an­cient ma­te­rial cleic acid po­ly­mer­ases, helicases, integrases, and other en­ from which vi­ral nu­cleic ac­ids can be re­cov­ered, might ap­ zymes. For ex­am­ple, as dis­cussed above, the RNA-dependent pear to make the or­i­gin of vi­ral ge­nomes an im­pen­e­tra­ble RNA po­ly­mer­ase is the only vi­ral hall­mark pro­tein con­served mys­tery. The oldest vi­ruses re­cov­ered from en­v i­ron­men­tal in RNA vi­ruses. Examination of the se­quences of vi­ral cap­sid sam­ples, the 30,000-year-old Pithovirus sibericum and Mol­ pro­teins re­veals at least 20 dis­tinct va­ri­e­ties that were de­rived livirus sibericum, iso­lated from Late Pleis­to­cene Si­be­rian from un­re­lated genes in an­ces­tral cells on mul­ti­ple oc­ca­sions. per­ma­frost, are sim­ply too rare and too young to pro­v ide The emerg­ing ev­i­dence there­fore sug­gests that vi­ral rep­li­ca­ much in­for­ma­tion on vi­ral evo­lu­tion. However, the dis­cov­ery tion en­zymes arose from precellular self-rep­li­cat­ing ge­netic of frag­ments of vi­ral nu­cleic ac­ids in­te­grated into host ge­ el­e­ments, while cap­sid pro­tein genes were cap­tured from un­ nomes, cou­pled with the ad­vances in de­ter­min­ing ge­nome re­lated genes in cel­lu­lar hosts. se­quences of vi­ruses and their hosts, has pro­v ided an im­ The com­po­si­tions of the eu ­kary­otic and bac­te­rial viromes proved un­der­stand­ing of the evo­lu­tion­ary his­tory of vi­ruses, dif­fer sub­stan­t ially (Chapter 1, Fig. 1.13). In bac­te­ria, most a topic dis­cussed in depth in Volume II, Chapter 10. known vi­r uses pos­sess dsDNA ge­nomes; fewer vi­r uses have How vi­ruses with DNA or RNA ge­nomes arose is a com­pel­ ssDNA ge­nomes, and there is a very lim­ited num­ber of vi­ ling ques­tion. A pre­dom­i­nant hy­poth­e­sis is that RNA vi­ruses ruses with RNA ge­nomes. In eu­k ary­otes, most of the vi­ are rel­ics of the “RNA world,” a pe­riod pop­u­lated only by RNA rome di­ver­sity is ac­counted for by RNA vi­r uses, but ssDNA mol­e­cules that cat­a­lyzed their own rep­li­ca­tion in the ab­sence and dsDNA vi­r uses are com­mon (Chapter 1, Fig. 1.13). The of pro­teins. During this time, bil­li­ons of years ago, cel­lu­lar life rea­sons for this dif­fer­ence are un­clear, but one pos­si­bil­ity could have evolved from RNA, and the ear­li­est cel­lu­lar or­gan­ is that the for­ma­tion of the eu­k ary­otic nu­cleus erected a 74 Chapter 3 bar­rier for DNA vi­r us re­pro­duc­t ion. On the other hand, the strand RNA ge­nomes (see Chapter 6). There is some ev­i­dence eu ­k ary­otic cy ­to­plasm with its ex­ten­sive mem­bra­nous sys­ that seg­mented RNA ge­ nomes might have arisen from tem might have been a hos­pi­ta­ble lo­ca­tion for RNA vi­r us monopartite ge­nomes, per­haps to al­low reg­u­la­tion of the pro­ rep­li­ca­t ion. duc­tion of in­di­vid­ual pro­teins (Box 3.5). Segmentation prob­a­ Viral ge­nomes dis­play a greater di­ver­sity of ge­nome com­ bly did not emerge to in­crease ge­nome size, as the larg­est RNA po­si­tion, struc­ture, and re­pro­duc­tion than any or­gan­ism. Un­ ge­nomes are monopartite. derstanding the func­tion of such di­ver­sity is an in­trigu­ing goal. As vi­ral ge­nomes are sur­vi­vors of con­stant se­lec­tive pres­sure, all­ con­fig­u­ra­tions must pro­vide ben­e­fits. One pos­si­ Genetic Analysis of Viruses bil­ity is that dif­fer­ent ge­nome con­fig­u­ra­tions al­low unique The ap­pli­ca­tion of ge­netic meth­ods to study the struc­ture and mech­a­nisms for con­trol of gene ex­pres­sion. These mech­a­ func­tion of an­i­mal vi­ral genes and pro­teins be­gan with de­vel­ nisms in­clude syn­the­sis of a polyprotein from (+) strand RNA op­ment of the plaque as­say by Renato Dulbecco in 1952. This ge­ nomes or pro­ duc­tion of subgenomic mRNAs from (−) as­say per­mit­ted the prep­a­ra­tion of clonal stocks of vi­rus, the B OX 3.5 E X P E R I M E N T S Origin of seg­mented RNA vi­rus ge­nomes Segmented ge­nomes are plen­ti­ful in the RNA point mu­ta­t ions that gave the RNAs a fit­ness 1 5’ AnAOH3’ vi­rus world. They are found in vi­rus par­ti­cles ad­van­tage over the stan­dard RNA arose be­ NSP1 from dif­fer­ ent fam­ i­ lies and can be dou­ ble fore frag­men­ta­tion oc­curred, im­ply­ing that (flavivirus NS5-like) stranded (Reoviridae) or sin­gle stranded, with the changes needed to oc­cur in a spe­cific se­ (+) (Closteroviridae) or (−) (Orthomyxoviridae) quence. The au­thors of the study con­clude: po­lar­ity. Some ex­per­i­men­tal find­ings sug­gest “Thus, ex­plo­ra­t ion of se­quence space by a vi­ 2 5’ AnAOH3’ that monopartite vi­ral ge­nomes emerged first ral ge­ nome (in this case an un­ seg­mented VP1 and then later frag­mented to form seg­mented RNA) can reach a point of the space in which ge­nomes. a to­tally dif­fer­ent ge­nome struc­ture (in this Insight into how such seg­mented ge­nomes case, a seg­mented RNA) is fa­vored over the 3 5’ AnAOH3’ may have been formed comes from stud­ies form that per­formed the ex­plo­ra­t ion.” While NSP2 with the pi­cor­na­v i­rus foot-and-mouth dis­ the frag­men­ta­t ion of the foot-and-mouth dis­ (flavivirus NS2b-NS3-like) ease vi­r us. The ge­nome of this vi­rus is a sin­gle ease vi­rus ge­nome may rep­re­sent a step on the mol­e­cule of (+) strand RNA. Serial pas­sage of path to seg­men­ta­tion, its rel­e­vance to what the vi­r us in baby ham­ster kid­ney cells led to oc­curs in na­ture is un­clear, be­cause the re­ 4 5’ AnAOH3’ the emer­gence of ge­nomes with two dif­fer­ent sults were ob­tained in cells in cul­ture. VP2, VP3 large de­le­tions (417 and 999 nu­cle­o­tides) in A com­pel­ling pic­ture of the gen­e­sis of a RNA ge­nome of JMTV vi­rus. The vi­ral ge­nome the cod­ing re­gion. Neither mu­tant ge­nome is seg­mented RNA ge­nome comes from the dis­ com­prises four seg­ments of sin­gle-stranded, (+) in­fec­tious, but when they are in­tro­duced to­ cov­ery of a new tick-borne vi­rus in China, sense RNA. Proteins en­coded by each RNA are in­ gether into cells, an in­fec­tious vi­rus pop­u ­la­ Jingmen tick vi­rus. The ge­nome of this vi­rus di­cated. RNA seg­ments 1 and 3 en­code fla­vi­vi­rus- tion is pro­duced. This pop­u ­la­tion com­prises a com­prises four seg­ments of (+) strand RNA. like pro­teins. mix­ture of each of the two mu­tant ge­nomes Two of the RNA seg­ments have no known se­ pack­aged sep­a ­rately into vi­rus par­ti­cles. In­ quence ho­mo­logs, while the other two are re­ an­other. Next, co­in­fec­tion of this seg­mented fection is suc­cess­f ul be­cause of com­ple­men­ta­ lated to se­quences of fla­v i­v i­ruses. The RNA fla­vi­vi­rus with an­other un­iden­ti­fied vi­rus could tion: when a host cell is in­fected with both ge­nome of fla­v i­v i­ruses is not seg­ment­ed: it is a have pro­duced the pre­cur­sor of Jingmen tick par­ti­cles, each ge­nome pro­v i­des the pro­teins sin­gle strand of (+) sense RNA. The pro­teins vi­rus. miss­ing in the oth­er. en­coded by RNA seg­ments 1 and 3 are non­ The re­sults pro­v ide new clues about the or­ Further study of the de­leted vi­ral ge­nomes struc­tural pro­teins that are clearly re­lated to i­gins of seg­mented RNA vi­rus­es. re­vealed the pres­ence of point mu­ta­tions in the fla­v i­v i­rus NS5 and NS3 pro­teins. Mo­re­no E, Ojosnegros S, García-Arriaza J, Escarmís other re­gions of the ge­nome. These mu­ta­tions The ge­nome struc­ture of this vi­r us sug­ C, Do­min­go E, Perales C. 2014. Exploration of se­ had ac­cu­mu­lated be­fore the de­le­tions ap­ gests that at some point in the past a fla­v i­v i­ quence space as the ba­sis of vi­ral RNA ge­nome seg­ peared and in­creased the fit­ness of the de­leted rus ge­nome frag­mented to pro­duce the RNA men­ta­t ion. Proc Natl Acad Sci U S A 111:6678–6683. ge­nome com­ pared with the wild-type ge­ seg­ments en­cod­ing the NS3- and NS5-like Qin XC, Shi M, Tian JH, Lin XD, Gao DY, He JR, nome. pro­teins. This frag­men­t a­t ion might have ini­ Wang JB, Li CX, Kang YJ, Yu B, Zhou DJ, Xu J, Ply- usnin A, Holmes EC, Zhang YZ. 2014. A tick-borne These re­sults show how monopartite vi­ral tially taken place as shown for foot-and-mouth seg­mented RNA vi­r us con­tains ge­nome seg­ments de­ RNAs may be di­v ided, pos­si­bly a path­way to a dis­ease vi­r us in cells in cul­t ure, by fix­ing of rived from un­seg­mented vi­ral an­ces­tors. Proc Natl seg­mented ge­nome. It is in­ter­est­ing that the de­le­tion mu­ta­tions that com­ple­mented one Acad Sci U S A 111:6744–6749.  Genomes and Genetics 75 mea­sure­ment of vi­rus ti­ters, and a con­ve­nient sys­tem for prac­t ice, cells are co­i n­fected with two mu­tants, and the fre­ study­ing vi­ruses with con­di­tional le­thal mu­ta­tions. Although quency of re­com­bi­na­t ion is cal­cu ­lated by di­v id­i ng the ti­ter a lim­ited rep­er­toire of clas­si­cal ge­netic meth­ods was avail­­able, of phe­no­t yp­i­cally wild-type vi­r us (Box 3.7) ob­tained un­der the mu­tants that were iso­lated (Box 3.6) were in­valu­able in re­stric­t ive con­d i­t ions (e.g., high tem­per­a­t ure) by the ti­ter elu­ci­dat­ing many as­pects of in­fec­tious cy­cles and cell trans­ mea­sured un­der per­mis­sive con­d i­t ions (e.g., low tem­per­a­ for­ma­tion. Contemporary meth­ods of ge­netic anal­y­sis based ture). The re­com­bi­na­tion fre­quency be­tween pairs of mu­ on re­com­bi­nant DNA tech­nol­ogy con­fer an es­sen­tially un­ tants is de­ter­mined, al­low­i ng the mu­ta­t ions to be placed on lim­ited scope for ge­netic ma­nip­u­la­tion; in prin­ci­ple, any vi­ral a con­t ig­u­ous map. Although a lo­ca­t ion can be as­signed for gene of in­ter­est can be mu­tated, and the pre­cise na­ture of the each mu­ta­t ion rel­a­t ive to oth­ers, this ap­proach does not re­ mu­ta­tion can be pre­de­ter­mined by the in­ves­ti­ga­tor. Much of sult in a phys­i­cal map of the ac­tual lo­ca­tion of the base change the large body of in­for­ma­tion about vi­ruses and their re­pro­ in the ge­nome. duc­tion that we now pos­sess can be at­trib­uted to the power of In the case of RNA vi­ruses with seg­mented ge­nomes, the these meth­ods. tech­nique of reassortment al­lows the as­sign­ment of mu­ta­ tions to spe­cific ge­nome seg­ments. When cells are co­in­fected with both mu­tant and wild-type vi­ruses, the prog­eny in­cludes Classical Genetic Methods reassortants that in­herit RNA seg­ments from ei­ther par­ent. Mapping Mutations The or­i­gins of the RNA seg­ments can be de­duced from their Before the advent of re­com­bi­nant DNA tech­nol­ogy, it was mi­gra­tion pat­terns dur­ing gel elec­tro­pho­re­sis (Fig. 3.11) or ex­tremely dif ­fi­cult for in­ves­ti­ga­tors to de­ter­mine the lo­ca­ by nu­cleic acid hy­brid­iza­tion. By an­a ­lyz­ing a panel of such tions of mu­ta­tions in vi­ral ge­nomes. The marker res­cue tech­ reassortants, the seg­ment re­spon­si­ble for the phe­no­t ype can nique (de­scribed in “Introducing Mutations into the Viral be iden­ti­fied. Genome” be­low) was a so­lu­t ion to this prob­lem, but be­fore it was de­vel­oped, other, less sat­is­fac­tory ap­proaches were Functional Analysis ex­ploit­ed. Complementation de­scribes the abil­ity of gene prod­ Recombination map­ping can be ap­plied to both DNA and ucts from two dif­fer­ent mu­tant vi­ruses to in­ter­act func­ RNA vi­ruses. Recombination re­sults in ge­netic ex­change tion­a lly in the same cell, per­m it­t ing vi­ral re­pro­duc­t ion. It be­t ween ge­nomes within the in­fected cell. The fre­quency of can be dis­t in­g uished from re­com­bi­na­t ion or reassortment re­com­bi­na­t ion be­t ween two mu­ta­t ions in a lin­ear ge­nome by ex­am­in­ing the prog­eny pro­duced by co­in­fected cells. True in­creases with the phys­i­cal dis­tance sep­a­rat­i ng them. In com­ple­men­ta­t ion yields only the two pa­ren­tal mu­tants, B OX 3.6 M E T H O D S Spontaneous and in­duced mu­ta­tions In the early days of ex­per­i­men­tal vi­rol­ogy, mu­ The low spon­ta­ne­ous mu­ta­tion rate of DNA tant vi­ruses could be iso­lated only by screen­ing vi­ruses ne­ces­si­tated ran­dom mu­ta­gen­e­sis by stocks for in­ter­est­ing phe­no­types, for none of ex­po­sure to a chem­i­cal mu­ta­gen. Mutagens the tools that we now take for granted, such as such as ni­trous acid, hy­drox­yl­amine, and al­kyl­ re­stric­tion en­do­nu­cle­ases, ef­fi­cient DNA se­ at­ing agents chem­i­cally mod­ify the nu­cleic acid quenc­ing meth­ods, and mo­lec­u­lar clon­ing in prep­a­ra­tions of vi­rus par­ti­cles, re­sult­ing in pro­ce­dures, were de­vel­oped un­til the mid to changes in base-pair­ing dur­ing sub­se­quent ge­ late 1970s. RNA vi­rus stocks usu­ally con­tain a nome rep­li­ca­tion. Base an­a­logs, in­ter­ca­lat­ing vi­a­bil­ity of the vi­rus. Virus mu­tants with this high pro­por­tion of mu­tants, and it is only a agents, or UV light are ap­plied to the in­fected phe­no­type re­pro­duce well at low tem­per­a­tures, mat­ter of de­vis­ing the ap­pro­pri­ate se­lec­tion cell to cause changes in the vi­ral ge­nome dur­ but poorly or not at all­at high tem­per­a­tures. con­di­tions (e.g., high or low tem­per­a­ture or ing rep­li­ca­tion. Such agents in­tro­duce mu­ta­ The per­mis­sive and non­per­mis­sive tem­per­a­ ex­po­sure to drugs that in­hibit vi­ral re­pro­duc­ tions more or less at ran­dom. Some mu­ta­tions tures are typ­i­cally 33 and 39°C, re­spec­tively, tion) to se­lect mu­tants with the de­sired phe­no­ are le­thal un­der all­ con­di­tions, while oth­ers for vi­ruses that rep­li­cate in mam­ma­lian cells. type from the to­tal pop­u­la­tion. For ex­am­ple, have no ef­fect and are said to be si­lent. Other com­ monly sought phe­ no­types are the live at­ten­u­ated po­lio­vi­rus vac­cine strains To fa­cil­i­tate iden­ti­fi­ca­tion of mu­tants, the changes in plaque size or mor­phol­ogy, drug re­ de­vel­oped by Al­bert Sabin are mu­tants that pop­u­la­tion must be screened for a phe­no­type sis­tance, an­ti­body re­sis­tance, and host range were se­lected from a vir­u­lent vi­rus stock (Vol­ that can be iden­ti­fied eas­ily in a plaque as­say. (that is, loss of the abil­ity to re­pro­duce in cer­ ume II, Fig. 7.11). One such phe­no­type is tem­per­a­ture-sensitive tain hosts or host cells). 76 Chapter 3 B OX 3.7 T E R M I N O L O G Y What is wild type? Terminology can be con­fus­ing. Virologists of po­lio­v i­rus ob­tained in 1909 un­doubt­edly is names of spe­cies (which are con­structs that as­ of­ten use terms such as “strains,” “var­i­a nts,” very dif­fer­ent from that of the vi­rus we call sist in the cata­log­ing of vi­ruses). A spe­cies and “mu­t ants” to des­ig­nate a vi­r us that dif­ wild type to­day. We dis­t in­g uish care­f ully be­ name is writ­ten in ital­ics with the first word fers in some her­i­t a­ble way from a pa­ren­t al or tween lab­o­ra­tory wild types and new vi­rus be­gin­ning with a cap­i­tal let­ter (other words wild-type vi­r us. In con­ven­tional us­age, the iso­lates from the nat­u­ral host. The lat­ter are should be cap­i­tal­ized if they are proper nouns). wild type is de­fi ned as the orig­i­nal (of­ten called field iso­lates or clin­i­cal iso­lates. For ex­am­ple, the caus­a­tive agents of po­lio­my­ lab­o­ra­to­r y-adapted) vi­r us from which mu­ The field of vi­ral tax­on­omy has its own eli­tis, po­lio­vi­rus types 1, 2, and 3, are mem­bers tants are se­lected and which is used as the nam­ing con­ven­tions which can cause some of the spe­cies Enterovirus C. A vi­rus name ba­sis for com­par­i­son. A wild-type vi­r us may con­f u­sion. Viruses are clas­si­fied into or­ders, should never be ital­i­cized, even when it in­ not be iden­t i­c al to a vi­r us iso­lated from na­ fam­i ­l ies, sub­fam­i ­l ies, gen­era, and spe­cies. cludes the name of a host spe­cies or ge­nus, and ture. In fact, the ge­nome of a wild-type vi­r us These names are al­ways ital­i­cized and start should be writ­ten in low­er­case: for ex­am­ple, may in­clude nu­mer­ous mu­t a­t ions ac­c u­mu­ with a cap­i­tal let­ter (e.g., Picornaviridae). To Sida ciliaris golden mo­saic vi­rus. A good ex­er­ lated dur­i ng prop­a­ga­t ion in the lab­o­ra­tory. en­sure clar­ity, the names of vi­ruses (like po­lio­ cise would be to see how of­ten we have ac­ci­ For ex­a m­ple, the ge­nome of the first iso­late vi­rus) should be writ­ten dif­fer­ently from the den­tally vi­o­lated these rules in this text­book. while wild-type ge­nomes re­sult from re­com­bi­na­t ion or re­ will oc­cur. In this way, the mem­bers of col­lec­t ions of mu­ assortment. If the mu­ta­tions be­ing tested are in sep­a ­rate tants ob­tained by chem­i­c al mu­ta­gen­e­sis were ini­t ially or­ genes, each vi­r us is ­able to sup­ply a func­t ional gene prod­ ga­n ized into com­ple­men­ta­t ion groups de­fi n­i ng sep­a­rate uct, al­low­i ng both vi­r uses to be re­pro­duced. If the two vi­ vi­ral func­t ions. In prin­ci­ple, there can be as many com­ple­ ruses carry mu­ta­tions in the same gene, no re­pro­duc­tion men­t a­t ion groups as genes. A B L M L R3 M 1 1 2 2 3 3 4 4 5 5 6 6 L M R3 7 7 8 8 Figure 3.11 Reassortment of in­flu­enza vi­rus RNA seg­ments. (A) Progeny vi­ruses of cells that are co­in­fected with two in­flu­enza vi­r us strains, L and M, in­clude both par­ents and vi­ruses that de­rive RNA seg­ments from them. Recombinant R3 has in­her­ited seg­ment 2 from the L strain and the re­main­ing seven seg­ments from the M strain. (B) 32P-labeled in­flu­enza vi­rus RNAs were frac­t ion­ated in a po­ly­ acryl­a mide gel and de­tected by au­to­ra­di­og­ra­phy. Migration dif­fer­ences of pa­ren­tal vi­ral RNAs (M and L) per­mit­ted iden­ti­fi­ca­tion of the or­i­gin of RNA seg­ments in the prog­eny vi­rus R3. Panel B re­printed from Racaniello VR, Palese P. 1979. J Virol 29:361–373.  Genomes and Genetics 77 Engineering Mutations into Viral Genomes The com­plete ge­nomes of poly­oma­v i­r uses, pap­i l­lo­ma­v i­ Infectious DNA Clones ruses, and ad­e­no­vi­ruses can be cloned in plas­mid vec­tors, and Recombinant DNA tech­niques have made it pos­si­ble to in­ such DNA is in­fec­tious un­der ap­pro­pri­ate con­di­tions. The DNA tro­duce any kind of mu­ta­tion any­where in the ge­nome of ge­nomes of her­pes­vi­ruses and pox­vi­ruses are too large to in­sert most an­i­mal vi­ruses, whether that ge­nome com­prises DNA into con­ven­tional bac­te­rial plas­mid vec­tors, but they can be or RNA. The quin­tes­sen­tial tool in vi­rol­ogy to­day is the in­ cloned into vec­tors that ac­cept larger in­ser­tions (e.g., cosmids fec­tious DNA clone, a dsDNA copy of the vi­ral ge­nome that and bac­te­rial ar­ti­fi­cial chro­mo­somes). The plas­mids con­tain­ing is car­ried on a bac­te­rial vec­tor such as a plas­mid. Infectious such cloned her­pes­v i­rus ge­nomes are in­fec­tious. In con­trast, DNA clones, or in vi­tro tran­scripts de­rived from them, can be pox ­v i­rus DNA is not in­fec­tious, be­cause the vi­ral pro­mot­ers in­tro­duced into cul­tured cells by trans­fec­tion (Box 3.8) to re­ can­not be rec­og­nized by cel­lu­lar DNA-dependent RNA po­ly­ cover in­fec­tious vi­rus. This ap­proach is a mod­ern val­i­da­tion mer­ase. Poxvirus DNA is in­fec­tious when early func­tions (vi­ral of the Hershey-Chase ex­per­i­ment de­scribed in Chapter 1. DNA-dependent RNA po­ly­mer­ase and tran­scrip­tion pro­teins) The avail­abil­ity of site-specific bac­te­rial re­stric­tion en­do­nu­ are pro­vided by com­ple­men­ta­tion with a helper vi­rus. cle­ases, DNA li­gases, and an ar­ray of meth­ods for mu­ta­gen­e­ RNA vi­rus­es. (i) (+) strand RNA vi­rus­es. The ge­no­mic sis has made it pos­si­ble to ma­nip­u­late these in­fec­tious clones RNA of ret­ro­vi­ruses is cop­ied into dsDNA by re­verse tran­scrip­ at will. Infectious DNA clones also pro­v ide a sta­ble re­pos­i­ tase early dur­ing in­fec­tion, a pro­cess de­scribed in Chapter 10. tory of the vi­ral ge­nome, a par­tic­u ­larly im­por­tant ad­van­tage Such DNA is in­fec­tious when in­tro­duced into cells, as are for vac­cine strains. As ol­i­go­nu­cle­o­tide syn­t he­sis has be­come mo­lec­u­larly cloned forms in­serted into bac­te­rial plas­mids. more ef ­fi­cient and less costly, the as­sem­bly of vi­ral DNA ge­ Infectious DNA clones have been con­structed for many nomes up to 212 kbp has be­come pos­si­ble (Box 3.9). (+) strand RNA vi­ruses. An ex­am­ple is the in­tro­duc­tion of a plas­mid con­tain­ing cloned po­lio­v i­rus DNA into cul­tured DNA vi­rus­es. Current ge­netic meth­ods for the study of mam­ma­lian cells, which leads to the pro­duc­tion of prog­eny most vi­r uses with DNA ge­nomes are based on the in­fec­tiv­ity vi­rus (Fig. 3.12A). The mech­a­nism by which cloned po­lio­v i­ of vi­ral DNA. When deproteinized vi­ral DNA mol­e­cules are rus DNA ini­ti­ates in­fec­tion is not known, but it has been sug­ in­tro­duced into per­mis­sive cells by trans­fec­tion, they gen­er­ gested that the DNA en­ters the nu­cleus, where it is tran­scribed ally ini­ti­ate a com­plete in­fec­tious cy­cle, al­t hough the in­fec­ by cel­lu­lar DNA-dependent RNA po­ly­mer­ase from cryp­tic, tiv­ity (num­ber of plaques per mi­cro­gram of DNA) may be pr