Phage replicase

Q P phage replicase ()X174 phage A protein R17 phage coat protein ribosomal protein SI 2 ribosomal protein LIO trpE... 

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 ... 

Figure 27. Detailed in vitro mechanism of RNA replication by Q/ -replicase [59]. RNA grows exponentially as long as template concentration is below enzyme concentration. Growth rate becomes constant and hence RNA concentration rises linearly when template concentration exceeds that of enzyme, while, finally, at large template excess, rate decreases down to zero due to enzyme inhibition and template double-strand formation. In these in vitro experiments, Q -replicase is present as environmental factor. In vivo the enzyme is formed during the first 20 rain after infection of host cell followed by RNA replication during second half of infectious cycle. After about 40 min, about a thousand infectious phage particles per cell are released in burst. These thousand infectious particles usually are minor part of total burst size.

The somewhat larger phage QP contains a 4.2-kb RNA genome. One subimit of the QP replicase is encoded by the virus, but three bacterial proteins are needed to form the complete replicase. They are ribosomal protein SI and elongation factors EF-Tu and EF-Ts, proteins that normally fimction within E. coli in translation of mRNA (Chapfer 29). Their abilify to associate with RNA has been exploited by the phage for a quite different purpose. 

A search for a specific inhibitor of the action of RNA replicase (RNA dependent RNA polymerase) another point at which viral replication could be inhibited independently of host cell metabolism, led to the screening of KXX) compounds against bacteriophage Q/S RNA replicase with E. coli as host cell. Thirty active compounds were obtained of which (LXIV) was the most active. This inhibited phage multiplication in E. coli by 99 per cent compared with control at 10 pg/ml whereas host cell growth was only inhibited by 50 per cent [243]. These authors hopefully conclude thus, further studies may produce a universal anti-RNA virus drug without side effects . 

Although these results suggest that the 2° and 3° structure of the phage RNA may play an important role in regulating the translation of certain cistrons, it is quite probable that these restrictions only apply to the translation of the infecting RNA molecules. Nascent plus strands, synthesized from the template double-stranded RNA later in infection, are also translated, but seem to make more A protein and replicase than mature RNA (Robertson and Lodish, 1970). This might, of course, be expected if the incomplete nascent chains are in a more extended configuration. 

At this point we mi t note that the replicase of the small RNA-containing phage Q3 synthesizes in vitro a mixture of plus and minus strands when synthesis is carried out in the presence of a host factor. In its absence, however, the same enzyme seems to be unable to use the plus strand as a proper template (SO). Althou the above reported experiments were purposely performed... 

Hie replicase of virus Q() (an RNA-phage) consists of four subunits one is encoded by the vM genome, wliile tlie other three are the host proteins EFTu, EFTs and R.p. SI. The Fig. shows a three dimensional model for the arrangement of R.p. in the SOS-subunit. Many antibiotics act by combining with R.p., e.g. streptomycin interacts with S12, and bacterial mutants resistant to streptomycin have been shown to liave an altered S12. Erythromycin interacts with L22, spiramycin with LA. 

RNA-synthetase (or RNA-replicase) formed by phage MS2 has been isolated in a purified form (Haruno et al., 1963). This enzyme possessed definite substrate specificity relative to phage RNA and was inactive in systems containing sRNA or ribosomal RNA as primer. 

Fig. 15. RNA-phage infection of a bacterial cell as a simple hyper-cyclic process. Using the translation machinery (T) of the host cell the infectious plus strand (I ) first instructs the synthesis of a protein subunit which associates with three host proteins to form a phage-specific RNA-replicase. This replicase complex (R) exclusively recognizes both phage-RNA strands, plus and minus, and replicates them.

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