Principles of Virology. Jane Flint

       Release of mRNA from rotavirus particles

      Rotaviruses, the most important cause of gastroenteritis in children, are large icosahedral viruses made of a three-shelled capsid containing 11 double-stranded RNA segments. The structure of this virus indicated that a large portion of the viral genome (~25%) is ordered within the particle and forms a dodecahedral structure (see Fig. 4.19). In this structure, the RNA molecules interact with the inner capsid layer and pack around the RNA polymerase located at the fivefold axis of symmetry. Further analysis of rotavirus particles in the process of synthesizing mRNA has shown that newly synthesized molecules are extruded from the capsid through several channels located at the fivefold axes (see the figure). Multiple mRNAs are released at the same time from such particles. On the basis of these observations, it has been suggested that each double-stranded genomic RNA segment is copied by an RNA polymerase located at a fivefold axis of symmetry. This model may explain why no double-stranded RNA virus with more than 12 genomic segments, the maximal number of fivefold axes, has been found.

      Three-dimensional visualization of mRNA release from rotavirus particles synthesizing mRNA. Structure of a rotavirus particle in the process of synthesizing mRNA. Parts of newly synthesized mRNA that are ordered, and therefore structurally visible, are shown in magenta at the fivefold axes of symmetry. The ordered portions of the mRNAs have been extended (gray tubes) to visualize exit from the capsid. Red circles depict mRNA cap structures. Courtesy of B. V. V. Prasad and Liya Hu, Baylor College of Medicine.

       Lawton JA, Estes MK, Prasad BV. 1997. Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. Nat Struct Biol 4:118–121.

Figure 6.25 Hepatitis delta virus RNA synthesis. (A) Schematic of the forms of hepatitis delta virus RNA and δ antigen found in infected cells. aa, amino acids; ORF, open reading frame. (B) Overview of hepatitis delta virus mRNA and genomic RNA synthesis. In steps 1 to 3, RNA synthesis is initiated by host RNA polymerase II at the indicated position on the (−) strand genomic RNA. The polymerase passes the poly(A) signal (purple box) and the self-cleavage domain (red circle). In steps 4 and 5, the 5′ portion of this RNA is processed by cellular enzymes to produce delta mRNA with a 3′ poly(A) tail, while RNA synthesis continues beyond the cleavage site and the RNA undergoes self-cleavage (step 6). RNA synthesis continues until at least one unit of the (−) strand genomic RNA template is copied. The poly(A) signal is ignored in this full-length (+) strand. In steps 7 to 10, after self-cleavage to release a full-length (+) strand, self-ligation produces a (+) strand circular RNA. In steps 11 to 20, mRNA synthesis initiates on the full-length (+) strand to produce (−) strands by a rolling-circle mechanism. Unit-length genomes are released by the viral ribozyme (step 15) and self-ligated to form (−) strand circular genomic RNAs. Data from Taylor JM. 1999. Curr Top Microbiol Immunol 239:107–122.

BOX 6.5

      BACKGROUND

       Ribozymes

      A ribozyme is an enzyme in which RNA, not protein, carries out catalysis. The first ribozyme discovered was the group I intron of the ciliate Tetrahymena thermophila. Other ribozymes have since been discovered, including RNase P of bacteria, group II self-splicing introns, hammerhead RNAs of viroids and satellite RNAs, and the ribozyme of hepatitis delta virus. These catalytic RNAs are very diverse in size, sequence, and the mechanism of catalysis. For example, the hepatitis delta virus ribozyme (see the figure) catalyzes a transesterification reaction that yields products with 2′,3′-cyclic phosphate and 5′-OH termini. Only an 85-nucleotide sequence is required for activity of this ribozyme, and can cleave optimally with as little as a single nucleotide 5′ to the site of cleavage.

      Ribozymes have been essential for producing infectious RNAs from cloned DNA copies of the genomes of (−) strand RNA viruses. Such transcripts often have extra sequences at the 3′ end. By joining the 85-nucleotide ribozyme fragment to upstream sequences, accurate 3′ ends of heterologous RNA transcripts synthesized in vitro can be obtained.

       Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. 1982. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31:147–157.

       Westhof E, Michel F. 1998. Ribozyme architectural diversity made visible. Science 282:251–252.

       Whelan SP, Ball LA, Barr JN, Wertz GT. 1995. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci USA 92:8388–8392.

      Crystal structure of the hepatitis delta virus ribozyme. The RNA backbone is shown as a ribbon. The two helical stacks are shown in red and blue, and unpaired nucleotides are gray. The 5′ nucleotide, which marks the active site, is green (PDB file 1cx0).

      All hepatitis delta virus RNAs are synthesized in the nucleus by DNA-dependent RNA polymerase II. The ability of this cellular enzyme to copy an RNA template provides a missing link in molecular evolution. This activity supports the hypothesis that an ancestor of RNA polymerase II copied RNA genomes that are thought to have existed during the ancient RNA world. During the course of evolution, enzymes with this property acquired the ability to copy DNA templates, facilitating the transition from RNA to DNA genomes. Today RNA polymerase II can still copy small RNAs such as the genome of hepatitis delta virus.

      The switch from mRNA synthesis to the production of full-length (+) strand RNA of hepatitis delta virus is controlled by suppression of a poly(A) signal. Full-length (−) and (+) strand RNAs are copied by a rolling-circle mechanism, and ribozyme self-cleavage releases linear monomers. Subsequent ligation of the two termini by the same ribozyme produces a monomeric circular RNA. The hepatitis delta virus ribozymes are therefore needed to process the intermediates of rolling-circle RNA replication. RNA polymerase II initiates viral mRNA synthesis at a position on the genome near the beginning of the delta antigen-coding region. Once the polymerase has moved past a polyadenylation signal and the self-cleavage domain (Fig. 6.25), the 3′ poly(A) of the mRNA is made by host cell enzymes. The RNA downstream of the poly(A) site is not degraded, in contrast to that of other mRNA precursors made by RNA polymerase II, but is elongated until a complete full-length (+) strand is made. The poly(A) addition site in this full-length (+) strand RNA is not used, perhaps because the delta antigen bound to the rod-like RNA blocks access of cellular enzymes to the poly(A) signal.

Do Ribosomes and RNA Polymerases Collide?

The genomic RNA of (+) strand viruses can be translated in the cell, and the translation products include the viral RNA polymerase. At a certain point in infection, the RNA polymerase copies the RNA in a 3′ → 5′ direction while ribosomes traverse it in an opposite direction (Fig. 6.26), raising the question of whether the viral polymerase avoids collisions with ribosomes. When ribosomes are frozen on polioviral RNA by using inhibitors of protein synthesis, replication is blocked. In contrast, when ribosomes are run off the template, replication of the RNA increases. These results suggest that ribosomes must be cleared from viral RNA before it can serve as a template for (−) strand RNA synthesis; in other words, replication and translation cannot