THE VIRAL REPLICASE

This finding suggests that the presence of VPg might be necessary to prime initiation of transcription of the nascent chains by the viral replicase. Lee at al. (20) suggested alternatively that VPg may play a role in the assembly of vimas... 

The only extensive studies which have been carried out on the nucleotide sequences of picornavims RNAs concern those sequences adjacent to the J - te miinal poly(A) tract (65-67). In view of their location, it might be expected that these sequences could be involved in specific interactions with the viral replicases, or else act in some other way as control elements. 

The 5 iid. of the nascent chains seems to be capped (63, 64) by a terminally-linked protein, covalently bound to the 5 end of the genomic RNA of picornaviruses (65-69). This led to the suggestion that the terminally-linked protein might be needed as a primer for the viral replicase (64, and for a more detailed discussion, see chapter 9)> t>nt there is not yet experimental evidence to support the notion of a direct involvement of this protein in the process of RNA replication. 

It was therefore proposed that either the viral polymerase is extremely instable and continuous protein synthesis is needed to restore the level of active enzymes (IO), or that proteolytic inactivation of the viral replicase leads to the decline of the rate of RNA synthesis observed late in the infectious cycle (81). 

Basically, in the absence of a better template, the viral replicase would bind to, and initiate the transcription of, the viral RNA. Conceivably, the very first step of this process would be the synthesis of the poly(U) tract (Figure 4> A), followed by the transcription of the heteropolymeric portion of the "plus" strand (Figure 4> But as soon as the first complementary strand is... 

Since the viral replicase has been recently solubilized and purified (see next chapter of this book), it will be soon possible to check the proposed model by readily designable experiments. 

Infection of HeLa cells with poliovirus elicits extensive proliferation of smooth cytoplasmic membranes (16). This occurs also in BHK cells infected with EMC virus. The results of an experiment in which smooth cytoplasmic membranes were isolated from aliquots of infected cells at various times after infection are shown in Figure 2. The membrane pellets were assayed for activity of EMC replicase and the protein and phospholipid content determined. For the first 4 hours of infection there is no change in the amount of celliilar smooth membranes. However, between 4 and 8 hours, at the time when the viral replicase is being formed, there occurs also a steep rise in the formation of smooth membranes. The protein content of the smooth membranes rises by about five-fold and the phospholipids by ten-fold. The diagram in Figure 5 depicts the increase of membrane material during infection and indicates also the changing ratios of protein to phospholipid which takes place in the smooth membranes synthesized between 4 and 8 hours after infection from 5.0 to 1.7. The low protein content of the new... 

Some viral protein synthesis (translation) must be occurring in the restrictive cells early in infection, since the viral replicase is required to initiate viral RNA replication and we can detect some replicase activity. Since some viral RNA synthesis does occur, cleavage of the viral polypeptide must have occurred... 

As noted, the viral RNA is of the plus (+) sense. Replicase synthesizes RNA of minus (-) sense using the infecting RNA as template. After minus RNA has been synthesized, plus RNA is made from this minus RNA. The newly made plus RNA strands now serve as messengers for virus protein synthesis. The gene for the maturation protein is at the 5 end of the RNA. Translation of the gene coding for the maturation protein (needed in only one copy per virus particle), occurs only from the newly formed plus-strand RNA as... 

One subunit (Mr 65,000) is the product of the replicase gene encoded by the viral RNA and has the active site for replication. The other three subunits are host proteins normally involved in host-cell protein synthesis the E. coli elongation factors Tu (Mt 30,000) and Ts (Mr 45,000)... 

These three host proteins may help the RNA replicase locate and bind to the 3 ends of the viral RNAs. 

RNA replicase isolated from Qj8-infected E. coli cells catalyzes the formation of an RNA complementary to the viral RNA, in a reaction equivalent to that catalyzed by DNA-dependent RNA polymerases. New RNA strand synthesis proceeds in the 5 —>3 direction by a chemical mechanism identical to that used in all other nucleic acid synthetic reactions that require a template. RNA replicase requires RNA as its template and will not function with DNA. It lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase. Unlike the DNA and RNA polymerases, RNA replicases are specific for the RNA of their own virus the RNAs of the host cell are generally not replicated. This explains how RNA viruses are preferentially replicated in the host cell, which contains many other types of RNA. 

RNA-dependent RNA polymerases, such as the replicases of RNA bacteriophages, are template-specific for the viral RNA. 

Coding versus Template Strands The RNA genome of phage Q/3 is the nontemplate or coding strand, and when introduced into the cell it functions as an mRNA. Suppose the RNA replicase of phage Q/3 synthesized primarily template-strand RNA and uniquely incorporated this, rather than nontemplate strands, into the viral particles. What would be the fate of the template strands when they entered a new cell What enzyme would such a template-strand virus need to include in the viral particles for successful invasion of a host cell ... 

Artificially enhanced error rates needed for the creation of sequence diversity in a population can be achieved readily with PCR. Reverse transcription and transcription are also susceptible to increase of mutation rates. These two and other new techniques for RNA amplification provide universal and efficient tools for the study of molecular evolution under laboratory conditions and make the usage of viral replicases with their undesirable sequence specificities obsolete. 

Am. A portion of the viral RNA contains information for making an enzyme, RNA replicase, which directs RNA synthesis from RNA. RNA replicase, synthesized in the host cell from viral RNA, directs the subsequent replication and transcription of viral RNA. 

It was suggested that poly(c) tracts mi t play a role in the replication of those viruses which contain it (28, 29) In particular, they speculated that such a poly(c) tract mi t function as a replicase-binding site, or a part of one, on the basis of two observations. First, Rosenberg at al. (39) reported that a partially purified EMC viral replicase preparation will synthesise poly(G) from a poly(c) template, but will not copy other synthetic primers. Second, the replicase of bacteriophage which has subunits of similar size to those of the EMCV replicase (39) can also specifically use poly(C) as a template for poly(G) synthesis. However, the significance of this specificity is unclear, since Q3... 

On the other hand the existence of non-coding regions involved, for example, in replicase recognition at either end of the RNA molecule would not be surprising. In addition it is not yet possible to exclude the possibility of still undetected protein especially if it were the product of a cistron which is translated at a lower frequency than the major one. This returns us to an important omission in our picornaviral map, the position of the viral "capping protein which is covalently bound to the of the picornaviral... 

Newly synthesized viral proteins including VPg (or a precursor of VPg - see below) may form the putative replicase, a protein complex capable of initiating RNA synthesis. In the process of initiation of RNA synthesis VPg may function as primer and be... 

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