Curso Biologa molecular 2011 Dep.

Bioqumica, Medicina, UASLP

REPLICACIN DE GENOMAS VIRALES Viral Genomes Come in a Variety of Forms and Can Be Either RNA or DNA
As discussed earlier, the DNA double helix has the advantages of stability and easy repair. If one polynucleotide chain is accidentally damaged, its complementary chain permits the damage to be readily corrected. This concern with repair, however, need not bother small viral chromosomes that contain only several thousand nucleotides. The chance of accidental damage is very small compared with the risk to a cell genome containing millions of nucleotides. The genetic information of a virus can, therefore, be carried in a variety of unusual forms, including RNA instead of DNA. A viral chromosome may be a single-stranded RNA chain, a double-stranded RNA helix, a circular single-stranded DNA chain, or a linear single-stranded DNA chain. Moreover, although some viral chromosomes are simple linear DNA double helices, circular DNA double helices and more complex linear DNA double helices are also common. Several viruses have protein molecules covalently attached to the 5' ends of their DNA strands, for example, and the DNA double helices from the very large poxviruses have their opposite strands at each end covalently joined through phosphodiester linkages. RNA viruses have particularly specialized requirements for replication, since to reproduce their genomes they must copy RNA molecules, which means polymerizing nucleoside triphosphates on an RNA template. Cells normally do not have enzymes to carry out this reaction, so even the smallest RNA viruses must encode their own RNA-dependent polymerase enzymes in order to replicate.

Both RNA Viruses and DNA Viruses Replicate Through the Formation of Complementary Strands
Like DNA replication, the replication of the genomes of RNA viruses occurs through the formation of complementary strands. For most RNA viruses this process is catalyzed by specific RNAdependent RNA polymerase enzymes (replicases). These enzymes are encoded by the viral RNA chromosome and are often incorporated into the progeny virus particles, so that upon entry of the virus into a cell, they can immediately begin replicating the viral RNA. Replicases are always packaged into the capsid of the so-called negative-strand RNA viruses, such as influenza or vesicular stomatitis virus. Negative-strand viruses are so called because the infecting single strand does not code for protein; instead its complementary strand carries the coding sequences. Thus the infecting strand remains impotent without a preformed replicase. In contrast, the viral RNA of positive-strand RNA viruses, such as poliovirus, can serve as mRNA and produce a replicase once it enters the cell; therefore the naked genome itself is infectious. The synthesis of viral RNA always begins at the 3' end of the RNA template, starting with the synthesis of the 5' end of the new viral RNA molecule and progressing in the 5'-to-3' direction until the 5' end of the template is reached. There are no error-correcting mechanisms for viral RNA synthesis, and error rates are similar to those in DNA transcription (about 1 error in 104 nucleotides synthesized). This is not a serious deficiency as long as the RNA chromosome is relatively short; for this reason the genomes of all RNA viruses are small relative to those of the large DNA viruses. All DNA viruses begin their replication at a replication origin, where special initiator proteins bind and then attract the replication enzymes of the host cell (see Figure 8-34). There are many different replication pathways, however. The complexity of these diverse replication schemes reflects, in part, the problem of replicating the ends of a simple linear DNA molecule, given a DNA polymerase enzyme that cannot begin synthesis without a primer . DNA viruses have solved this problem in a variety of ways: some have circular DNA genomes and thus no ends; others have linear DNA genomes that repeat their terminal sequences or end in loops; while still others have special terminal proteins that serve to prime the DNA polymerase directly.

Viruses Exploit the Intracellular Traffic Machinery of their Host Cells

All viruses have only a limited amount of nucleic acid in their genome, and so they must parasitize host-cell pathways for most of the steps in their reproduction. In fact, because viral products are usually synthesized in large amounts during infection, and because during its life cycle the virus follows a sequential route through the compartments of the host cell, virus-infectedcells have served as important models for tracing the pathways of intracellular transport and for studying how essential biosynthetic reactions are compartmentalized in eucaryotic cells. Enveloped animal viruses, in which the genome is enclosed in a lipid-bilayer membrane, have exploited the compartmentalization of the cell to an especially fine degree. To follow the life cycle of an enveloped virus is to take a tour through the cell. A well-studied example is Semliki forest virus, which consists of a single-stranded RNA genome surrounded by a capsid formed by a regularly arranged icosahedral (20-faced) shell composed of many copies of one protein (called C protein). The nucleocapsid (genome + capsid) is surrounded by a closely apposed lipid bilayer that contains only three types of polypeptide chains, each encoded by the viral RNA. These envelope proteins form heterotrimers that span the lipid bilayer and interact with the C protein of the nucleocapsid, linking the membrane and nucleocapsid together (Figure 6-77). The glycosylated portions of the envelope proteins are always on the outside of the lipid bilayer, and each trimer forms a "spike" that can be seen in electron micrographs projecting outward from the surface of the virus (Figure 6-77C). Infection is initiated when an envelope protein on the virus binds to a normal cell protein that serves as its receptor on the host-cell plasma membrane. The virus then uses the cell's normal endocytic pathway to enter the cell by receptor-mediated endocytosis and is delivered to early endosomes (discussed in Chapter 13). But instead of being transferred from endosomes to lysosomes, the virus escapes from the endosome by virtue of the special properties of one of its envelope proteins. At the acidic pH of the endosome, this protein causes the viral envelope to fuse with the endosome membrane, releasing the bare nucleocapsid into the cytosol. The nucleocapsid is "uncoated" in the cytosol, releasing the viral RNA, which is then translated by host-cell ribosomes to produce a virus-encoded RNA polymerase. This in turn makes many copies of viral RNA, some of which serve as mRNA molecules to direct the synthesis of the structural proteins of the virus - the capsid C protein and the three envelope proteins. The newly synthesized capsid and envelope proteins follow separate pathways through the cytoplasm. The envelope proteins, like the plasma membrane proteins of the host cell, are synthesized by ribosomes that are bound to the rough ER; in contrast, the capsid protein, like the cytosolic proteins of the cell, is synthesized by ribosomes that are not membrane bound. The newly synthesized capsid protein binds to the recently replicated viral RNA to form new nucleocapsids. The envelope proteins, in contrast, are inserted into the membrane of the ER, where they are glycosylated, transported to the Golgi apparatus, and then delivered to the plasma membrane (Figure 6-78). The viral nucleocapsids and envelope proteins finally meet at the plasma membrane. As a result of a specific interaction with a cluster of envelope proteins, the nucleocapsid forms a bud whose envelope contains the envelope proteins embedded in host-cell lipids. Finally, the bud pinches off and a free virus is released on the outside of the cell. The clustering of envelope proteins as they assemble around the nucleocapsid during viral

RNA Tumor Viruses Are Retroviruses

For one group of RNA viruses, the so-called RNA tumor viruses, the infection of a permissive cell often leads simultaneously to a nonlethal release of progeny virus from the cell surface by budding and a permanent genetic change in the infected cell that makes it cancerous. How RNA virus infection could lead to a permanent genetic alteration was unclear until the discovery of the enzyme reverse transcriptase, which transcribes the infecting RNA chains of these viruses into complementary DNA molecules that integrate into the host cell genome. RNA tumor viruses -which include the first well-known tumor virus, the Rous sarcoma virus - are members of a large class of viruses known as retroviruses. These viruses are so named because as part of their normal life cycle they reverse the normal process in which DNA is transcribed into RNA. The enzyme reverse transcriptase is an unusual DNA polymerase that uses either RNA or DNA as a template (Figure 6-81); it is encoded by the retrovirus RNA and is packaged inside each viral capsid during the production of new virus particles. When the single-stranded RNA of the retrovirus enters a cell, the reverse transcriptase brought in with the capsid first makes a DNA copy of the RNA strand to form a DNA-RNA hybrid helix, which is then used by the same enzyme to make a double helix with two DNA strands. The two ends of the linear viral DNA molecule are recognized by a virus-encoded integrase that catalyzes the insertion of the viral DNA into virtually any site on a host-cell chromosome (see Figure 6-70). The next step in the infectious process is transcription of the integrated viral DNA by host-cell RNA polymerase, producing large numbers of viral RNA molecules identical to the original infecting genome. Finally, these RNA molecules are translated to produce the capsid, envelope, and reverse transcriptase proteins that are assembled with the RNA into new enveloped virus particles, which bud from the plasma membrane Both RNA and DNA tumor viruses transform cells because the permanent presence of the viral DNA in the cell causes the synthesis of new proteins that alter the control of host-cell proliferation. The genes that code for such proteins are called oncogenes. Unlike DNA tumor viruses, whose oncogenes typically encode normal viral proteins essential for viral multiplication, the oncogenes carried by RNA tumor viruses are modified versions of normal host-cell genes that are not required for viral replication. Since only a limited amount of RNA can be packed into the capsid of a retrovirus, the acquired oncogene sequences often replace an essential part of the retroviral genome. In Chapters 15 and 24 we discuss how viral oncogenes have provided important clues to the causes and nature of cancer, as well as to the normal mechanisms that control cell growth and division in multicellular animals. We also discuss how the random integration of viral DNA into genomes can alter normal genes and thereby affect cell behaviour.

Flow of events during the replication of picornaviruses The linear single-stranded RNA viruses form 3 groups. Picornaviruses and togaviruses are examples of the first group. These genomes have two functions . The first of these functions is to serve as a messenger RNA. By convention, viruses whose genomes can and do serve as messengers are known as plus (+) strand viruses. Following entry into the cell, picornavirus RNA binds to ribosomes and is translated in its entirety. The product of this translation - the polyprotein - is then cleaved by proteolytic enzymes. While secondary cleavages clearly involve virus-specified proteases, there is good evidence that the polyprotein itself is enzymatically active in trans, that is, each molecule cannot cleave itself but it can cleave other polyproteins. The second function of the genomic RNA is to serve as a template for the synthesis of a complementary (-) strand RNA by a polymerase derived from cleavage of the polyprotein. The (-) RNA strand then serves in turn as a template to make more (+) RNA strands. The progeny (+) strands can then serve as (a) mRNA or (b) templates to make more (-) RNA strands.

El virus de la polio tiene un IRES en su UTR5del ARN+. La RNA replicasa utiliza un primer VPg que es un pptido pequeo con un uracilo como cap viral. La proteasa viral degrada un fragmento de eIF4G y aumenta as el uso del IRES viral para su traduccin.

Rotavirus (dsRNA) segmentado

Virus de la Influenza (ssRNA-)

Flow of events during the replication of orthomyxoviruses and paramyxoviruses Orthomyxoviruses, paramyxoviruses, bunyaviruses, arenaviruses, and rhabdo-virusesCharacteristically, their genomic RNAs must serve two template functions, in the first step for the synthesis of mRNA, and in the second for the synthesis of complementary (+) strands which serve as a template to make viral progeny genomes.

Retrovirus

Flow of events during the replication of retroviruses

Characteristically, retrovirus genomes are monopartite, but diploid, and the two strands are either partially hydrogen-bonded to another macromolecule or base-paired in a fashion as yet unknown. Following infection, the sole known function of the genomic RNAs is to serve as a template for the synthesis of viral DNA. Inasmuch as eukaryotic cells lack enzymes competent to perform this function, the virion contains, in addition to the genome, an RNA-dependent DNA polymerase (reverse transcriptase) as well as a mixture of host transfer RNAs, one of which serves as a primer. The key steps in the genome transcription are (a) binding of the tRNA - reverse transcriptase complex to the genomic RNA, (b) synthesis of a DNA molecule complementary to the genomic RNA coupled with the digestion of the RNA by a viral ribonuclease (RNase H, also packaged in the virion) specific for RNA in RNA-DNA hybrids, and (c) synthesis of the complementary DNA strand and completion of a linear DNA molecule containing in its entirety the sequences contained in the genomic RNA, but with the duplication of two small sequences, one from the 3 terminus of the RNA duplicated at the 5 terminus of the DNA, and one from the 5 terminus of the RNA duplicated at the 3 terminus of the DNA

Virus de Hepatitis B

The hepadna viruses exemplified by hepatitis B virus. The DNA of this virus is first repaired and converted into a closed circular molecule by a DNA polymerase packaged in the virion, and then transcribed into two classes of RNA molecules, i.e. a mRNA specifying proteins and a genomic RNA which is transcribed by a reverse transcriptase to make the genomic DNA.