In this article we will discuss about the replication of genetic RNA.
Several viruses and bacteriophages contain single- or double-stranded RNA as their genetic material (Table 3.1).
Replication of these RNA chromosomes occurs in the host cytoplasm following one of the two modes given below:
(i) Direct use of RNA template for RNA synthesis (RNA RNA) by the enzyme –‘RNA replicase” or “RNA dependent RNA polymerase”.
(ii) Use of RNA template by the enzyme “reverse transcriptase” to produce a complementary DNA (cDNA) molecule, which then serves as the template for the synthesis of complementary RNA by the enzyme RNA polymerase (RNA à DNA à RNA).
(i) Direct replication of genetic RNA:
The enzyme RNA dependent RNA polymerase, also called “replicase” catalyzes the direct replication of genetic RNA. Spiegelman and his associates isolated one form of this enzyme from bacteriophage QB in 1965; it could function in the presence of an RNA template, Mg2+ ions and the four ribonucleotide triphosphates (ATP, GTP, CTP, UTP) in vitro.
Double stranded RNA replicates in a semiconservative manner similar to DNA to produce two double-stranded progeny molecules.
Single-stranded genetic RNA found in some viruses, such as, TMV is called the plus (+) strand RNA; it acts as a template for the synthesis of a complementary minus (-) strand to yield a double-stranded RNA molecule. This double-stranded RNA undergoes semiconservative replication like other double-stranded RNA molecules. However, only the plus (+) strand is packed into the virus.
(ii) RNA replication via complementary DNA (cDNA):
The oncogenic viral RNA is a single-stranded molecule of about TO kb with terminal repetition, and is designated as plus (+) strand. The virion contains two RNA molecules held together by a dimer linkage structure at the 5′-end, formed by tRNA (an uncharged host tRNA present in the virion).
The 3′-end of the tRNA has a sequence of 18 bases that pair to a site 100-200 bases from the 5′-end of one of the two viral RNA molecules. The other RNA molecule may also be paired near its 5′-end with the tRNA; thus both RNA molecules form a dimer. Upon denaturation, the RNA dimer separates into two identical molecules indicating that the virion is diploid, i.e., possesses two copies of the genome.
The retroviruses having single-stranded RNA contain about 30 molecules/virion of the enzyme “reverse transcriptase”. This enzyme is also called “RNA dependent DNA polymerase”; it was discovered in 1970 independently by Temin and Baltimore. It synthesizes DNA molecule complementary to the viral RNA.
An uncharged host tRNA present in the virion acts as the primer for DNA synthesis, using the genetic RNA strand as template. The DNA strand (- strand) so obtained is called complementary DNA (cDNA). This DNA synthesis is catalyzed by the enzyme reverse transcriptase. Synthesis of (+) strand of DNA complementary to the (-) strand DNA begins even before the synthesis of the (-) strand is completed.
The viral RNA is degraded and removed by the same enzyme reverse transcriptase as soon as the synthesis of (-) strand is completed. Reverse transcriptase has the 5’—>3′ exonuclease (RNAaseH) activity and degrades the RNA strands present in DNA-RNA hybrids, beginning from the 5′-end of the RNA (5’—>3′ exonuclease activity) strand.
The newly synthesized viral DNA is double-stranded with a continuous (-) strand and a discontinuous (+) strand. The gaps are filled and nicks are sealed by DNA ligase. This double stranded DNA moves to the nucleus where it becomes integrated into the host chromosome. It remains in the host genome as “provirus” and replicates with the host DNA.
The double-stranded DNA produced has a terminal redundancy which is absent in the viral RNA. This redundancy facilitates the circularization of the DNA molecule and persists in the integrated provirus. The viruses that use this reverse transcriptase-mediated pathway for viral integration into the host chromosome and replication are called retroviruses.
The replication proceeds as follows (Fig. 3.20):
(1) A tRNA (uncharged host tRNA) molecule already present in the virion, and paired to the 5′-end of the viral RNA acts as a primer for the synthesis of the (-) strand DNA, which is initiated close to the 5′-end of the viral RNA. The synthesis of this DNA strand is continuous and proceeds till the 5′-end, including the terminal repetitions, are reached.
(2) At this point, the synthesis of the DNA (-) strand stops; the DNA produced so far is called strong stop DNA which remains attached to the tRNA primer.
(3) The reverse transcriptase switches templates, carrying the nascent DNA with it to the new template. Thus the strong stop DNA separates from 5-end and “jumps” to the terminal repetition at 3′-end. In this reaction, the “R region” at the 5′ terminus of the RNA template is degraded by RNAaseH activity of reverse transcriptase.
(4) The removal of the “R region” at 5′-end allows the R region at 3′-end to base pair with the newly synthesized DNA (Fig. 3.20). As a result, a “U3 segment” is added to the 5′- end to make a stretch of sequence “5′ U5-R- U3 3″‘ which is called the long terminal repeat (LTR). Similar series of events produce “5’ U5-R-U3″‘, sequence at the 3’-end by adding a U5 segment).
(5) Synthesis of the DNA (-) strand continues in the 5’—>3′ direction till it reaches the 5′-end of RNA; this produces a DNA-RNA hybrid molecule.
(6) The viral RNA is degraded by the exonuclease (RNAase H) activity of the enzyme reverse transcriptase, beginning from the 5′-end.
(7) Synthesis of the DNA (+) strand begins at different sites along the (-) DNA strand which is used as the template. This synthesis is discontinuous, uses small RNA primers and yields several small (+) DNA fragments.
(8) The 3′-end of the (-) DNA strand again “jumps” to the other end (5′-end) where it pairs with the 5′-end of fragment of the (+) strand DNA, and the R-U3 region is synthesized.
(9) The fragments of the (+) strand DNA are joined by the polynucleotide ligase.
(10) The tRNA is removed and a double-stranded DNA is produced which is longer than the viral RNA with long terminal repeats (LTR) on both sides (Fig. 3.20).
(11) This DNA molecule may be circularized and can become integrated within the host chromosome.
The viral genome produces oncogenic proteins which transform the normal host cell into a tumor cell. The viral RNA is produced from the pro-viral DNA and combines with coat proteins to yield new viral particles. These viral particles are released and are enclosed by the host cell membrane. The life cycle of a retrovirus is shown in Fig. 3.21.