We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848. By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Albl, maps to the N gene. Sequencing of the Albl N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Albl mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.

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We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phageT7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and iysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 300. Positive complementation results in a 100-fold increase in phage titer.Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9-, 10-), within head structures present in an extract that contains no phage DNA (gene 5-). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5-,9, 10-), which complements gene 19-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.In recent years there has been rapid progress in our understanding of the mechanisms by which complex bacteriophages are assembled. Thus, for bacteriophage T4, we know a great deal about the pathways of assembly of tail fibers (1-4), baseplates, sheaths, and cores of the tail (5-9) and assembly of the head (10). These advances have been aided greatly by the development by Edgar and Wood of methods for performing these reactions in cell-free extracts (11,12).The DNA of several bacteriophages (T4, T7, X, and P22) has been shown to replicate as molecules that are several times longer than the DNA found inside the head of the mature virus (13)(14)(15)(16)(17). It is also known that maturation of these long chains of DNA to monomeric units is closely coupled to the formation of the phage head, since cells infected with mutants that are defective in head formation accumulate these long molecules of immature DNA (18-21).However, our knowledge of the molecular mechanisms whereby the immature phage DNA enters the head ("DNA packaging") and is cleaved to monomeric length ("DNA maturation") has been limited, in part, by the inability to perform these reactions in vitro. Recently, however, assays have been developed to demonstrate packaging of phage DNA in vitro. Kaiser and Masuda (22) showed that exogenous lambda DNA could be packaged by extracts from induced lysogens and Hohn and Hohn (23) have demonstrated that head-related particles containing no DNA ("petit lambda") can be filled with DNA in vitro. Finally, the experiments of Pruss, Goldstein, and Calendar (24) showed that the size of the head of phage P2 is determined solely by the proteins involved and not by the phage DNA.Bacteriophage T7 may be a convenient system in which to study DNA packaging and maturation because it is a 3545 relatively simple virus for which there is a great deal of information about its genetics and physiology (25). The DNA of phage T7 is a unique, terminall...

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of Escherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 106 between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.

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