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The adenovirus major late promoter (MLP) has played a preeminent role in the analysis of transcription initiation in mammalian cells, and is an outstanding example of the ways in which the study of adenovirus has led to fundamental insights into general cellular processes. The aim of this chapter is to give a comprehensive review of the structure and function of this model mammalian promoter. After a brief description of late transcription in the adenovirus replication cycle, the experimental evidence for the current consensus on the genetic structure of the MLP, including a consideration of non-primate adenovirus MLPs, will be reviewed. Next, the functions of the MLP in the viral life cycle will be examined, and some of the problems that remain to be resolved will be addressed. The review ends with some ideas on how the knowledge of the structure and function of the MLP can be used in designing virus vectors for specific experimental purposes.

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in Hin dIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between Eco RI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and Bam HI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202 Eco RI fragment A was crossed with the Ad5wt Bam HI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.

Individual colonies of an adenine-requiring strain of the genotype ade 2-1, SUPQ5, [psi minus] exhibit high and uniform frequencies of ADE+ revertants (5-1 times 10-5). The frequencies for strains of the genotypes ade 2-1, SUPQ5+, [psi+] and ade 2-1 SUPQ5+, [psi minus] are by contrast 1-2 times 10-7 and 0-5 times 10-7 respectively. Mutation from [psi minus] to [psi+] can occur. This suggests that the original mutation from [psi+] to [psi minus] is not necessarily a physical loss of, or extensive deletion, the extrachromosomal elements. Although mutation from [psi minus] to [psi+] occurs, the majority of ADE+ revertants from ade 2-1, SUPQ5, [psi minus] strains contain other types of mutation. Crosses between most ADE+ revertants and ade 2-1, SUPO5+, [psi minus] strains yield adenine-requiring diploids and tetrad segregations of 0:4, 1:3 and 2:2 ADE+ :ade minus. It is argued that most mutations give rise to weak recessive suppressors (SUPX) which are themselves incapable of suppressing the ochre allele ade 2-1, unless the allele SUPQ5 is also present. Tests with two suppressors, S-theta and S-zeta, isolated by Dr R. A. Gilmore, which are themselves incapable of suppressing ade 2-1, show that they do not do so even in the presence of SUPQ5. Thus the set of suppressors isolated by us is a special class which can perhaps only be observed in the genetic background ade 2-1, SUPQ5, [psi minus]. There is evidence that some of the new suppressors are capable, in the absence of SUPQ5, of weakly suppressing trp 5-48 and perhaps can 1-100. All ochre alleles are, however, suppressed by the combination SUPQ5, SUPX. SUPQ5 is able to suppress trp 5-48, his 5-2, lys 1-1 and can 1-100 in a [psi minus] background, although it does so variably. It is incapable of suppressing ade 2-1 in such a background. It is argued that the alteration in the [psi] determinant results in a lowered efficiency of suppression by SUPQ5 rather than a changed specificity.

Interferon induces protection of enucleated BSC-1 cells against infectious vesicular stomatitis virus production if cells are treated before, but not after, enucleation.