Search

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' untranslated regions (3' UTRs) of ORF10-targeted transcripts conferring sensitivity to export inhibition. The Rae1-ORF10 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

International audience; During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E), and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the transcription start site (TSS). Moreover, in the case of the β-and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies (β and γ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors-especially ICP4-play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein.

The late expression factor-8 gene (lef-8) of Autographa californica M nucleopolyhedrovirus encodes the largest subunit of the virally encoded DNA-directed RNA polymerase specific for the transcription of late and very late viral genes. The sequence of lef-8 predicts a C-terminal motif of 13 amino acids that is conserved in other polymerases. Detailed mutagenesis throughout lef-8 was performed, including this C-terminal motif, to define sequences required for late promoter activation. It was found that the conserved C-terminal motif was critical for late gene expression. In addition, regions throughout the entire lef-8-encoding sequence were important for optimal function, suggesting complex protein-protein and protein-DNA interrelationships in the late gene-specific viral transcriptosome.

This chapter concentrates on the mechanisms by which human papillomaviruses (HPVs) regulate viral transcription and replication during the productive life cycle. The first studies which examined papillomaviruses concentrated on the bovine papillomaviruses (BPVs) with the hope that BPV-1 would serve as a useful model for the high-risk human viruses. While many similarities exist between the two viral types, a significant number of differences are also present. For this reason, the author concentrates the discussion on the HPV types and refers to BPVs only when no information is available in the human system. In order to understand how viral gene expression is regulated during a productive infection, it is first important to describe the transcripts which are synthesized. One of the most remarkable features of HPV transcription is that all early and late transcripts are polycistronic. The E1 and E2 proteins function in viral replication and may also modulate viral expression. A detailed discussion of the role of these proteins in replication is provided in the chapter. In order to further understand the interplay of these two viral functions, the chapter discusses the control of viral replication in detail. Given the tight linkage of late gene expression and amplification to epithelial differentiation, it is likely that cellular factors play important roles.

Early and late genes of human and animal papillomaviruses show a codon composition seemingly unfavorable for expression in mammalian cells. It remains unclear how the viruses manage to achieve high levels of late gene expression during the viral life cycle. One possible solution could be that the availability of certain t-RNAs changes with progressing stages of cellular differentiation. Previous studies have demonstrated that modification of codon usage of papillomavirus late (L1 and L2) and early genes (E7) can overcome poor expression of these proteins both in transient and in stable expression systems. This was shown not only for human but also for plant cells. Two strategies can be employed to alter codon usage: elimination of only those codons that are rarely used in a particular expression system, or exchange of all possible codons by the ones most frequently used. Currently, there are two protocols for codon modification--a template-less polymerase chain reaction (PCR)-based protocol, in which very long overlapping oligodeoxynucleotides are used in an overlap-extension reaction, or a ligase chain reaction, in which shorter oligodeoxynucleotides are fused together after an annealing procedure. Both methods are presented and discussed.

On account of their relatively small, defined genomes, viruses have long been a convenient model system in which to study eucaryotic gene expression. Usually, viral infection is followed by early gene expression, which allows the subsequent processes of viral DNA replication and late gene expression to proceed. All these processes must be precisely regulated and involve both virally encoded and host proteins.

The transcriptional characterization of the gene coding for the p12 attachment protein of the African swine fever virus is presented. The results obtained have been used to generate the first detailed transcriptional map of an African swine fever virus late gene. Novel experimental evidence indicating the existence of major differences between the mechanisms controlling the transcription of late genes in African swine fever virus and poxviruses is provided.

The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).

The lac transducing phage, lambda plac5, carries a segment of the E. coli lac operon on the left side of the b2 region of the lambda phage. In the absence of additional cyclic AMP, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. This phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the cI and N gene products; however, the repressive action of the cro gene product at high multiplicity of infection is not observed although some turn off at very late time is detected. To pin down this phage promoter, results described in this communication and those described elsewhere can rule out the promoter PI, PR, P'R, and the promoter PL also looks rather unlikely. No firm identification of this phage promoter has been made, but the promoter(s) in the b2 region (the b2 promoter) is proposed. The phage promoter responsible for beta-galacrosidase synthesis is shown to be a weak promoter, requires the Q gene product or one (or more) of the late gene products for activation, and the time of expression is very late.

The effect of multiplicity of infection was studied in Escherichia coli with lambda phage, using phage endolysin as an example of a late gene product. A very sensitive endolysin assay method was used so that the initiation time of endolysin synthesis could be more accurately determined. It was observed that high multiplicity of infection (1) increases the rate of lysogenization, (2) progressively delays lysis time, and (3) significantly delays and reduces the synthesis of endolysin in lamdacIII+ cII+ -infected cells. The extent of delay and reduction in endolysin synthesis increases with increasing multiplicity. In contrast, lamdacIII67cII68-infected cells show no delay in endolysin synthesis at high multiplicity of infection when compared with the lamdacIII+ cII+ -infected cells. The results suggest that (1) the expression of cIII and cII genes is multiplicity dependent, (2) high multiplicity of infection enhances the expression of the cIII and cII genes, and (3) the expression of the cIII and cII genes interferes with the expression of the late genes. A model to explain how the expression of the cIII and cII genes interferes with the expression of the late genes is proposed.

We have constructed deletion mutants of simian virus 40 (SV40) lacking the two tandemly repeated copies or all three copies of the 21-bp repeated sequence located in the origin region. The mutants were viable, but had lower infectivities compared to the wild type. The mutant lacking two copies of the 21-bp repeat grew fairly well indicating that the one copy of the 21-bp repeat it contains is adequate. The other mutant lacking all the three copies of the 21-bp repeat was also viable but grew poorly. The viability of this mutant suggests that the upstream 72-bp repeated sequence compensates, though only partially, for the absence of the 21-bp repeat. The growth deficiencies of the deletion mutants could not be overcome by complementation with temperature-sensitive helper mutants providing either the early or the late functions of the virus, suggesting that the deficiencies lie in both early and late gene expression and/or in replication.

Flowering in Pisum . Three distinct phenotypic classes determined by the interaction of a dominant early and a dominant late gene

Chorion genes in the silkmoth Bombyx mori are clustered in a small region of one chromosome. Genes are grouped within this locus according to their expression in either early, middle, or late stages of choriogenesis. The entire set of late genes and a large fraction of the middle genes have been cloned and extensively characterized. We report here the first molecular characterization of a set of early chorion genes clustered within a 22.5-kb region. Transcripts homologous to these genes appear in the very first choriogenic follicle but disappear with different kinetics. One of the three early genes, 5H4, is the first example of a chorion gene which is not a member of a multigene family, or divergently paired with another chorion gene. The other two early genes, (ErA.1 and ErB.1) are divergently paired with each other in the manner of the middle and late chorion genes. The common 5′ region between ERA.1 and ErB.1 is significantly shorter and does not contain certain sequence elements shared by the highly conserved 5′ regions of middle and late chorion gene pairs. This evidence taken together with the fact that 5H4 is unpaired indicates that the early genes may utilize a regulatory mechanism somewhat different from the middle and late gene families.

A rtrp/lac W205 substitution, fused to the late ragion of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q + and Q - phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ is only significantly active when supplied in cis to its site of action.

The late genes of the temperate Salmonella phage P22 comprise an operon. The late operon of P22 contains within it a gene ( 9 ) which is separated from the other late genes by a set of genes ( imm I) which are regulated independently of the late genes. These results were obtained by exploiting the orientation-dependent polarity of insertions of the translocatable ampicillin-resistance element Tn1. The data are consistent with a model of P22 late gene regulation in which the positive activator of the late operon, gene 23 , can act as a selective antagonist of transcription termination.

We have examined the organization and properties of the genes around the region controlling late gene expression in phage 82. We have isolated mutants of λ(QSR)82 hybrid phages that are analogous to λ Q−, S−, and R− mutants; they map on the 82 genome in the same order as the corresponding λ mutations. The QSR region of 82 encodes a small, r-strand-specific transcript which is probably the 82 analog of the λ 6 S RNA, and thus may be involved in the regulation of late gene expression in 82.

Eleven cistrons of genes active in lytic growth of phage P22 were classified by phenotypic expression of their amber mutants in nonpermissive Salmonella typhimurium. Seven cistrons code for late functions according to their lysis positive phenotype. Of the remaining four cistrons one codes for lysozym synthesis, two for phage directed DNA synthesis and one seems to be engaged in regulation of late gene expression.

The interactions between bacteriophage P 2 and its satellite phage P 4 comprise a promising model system for studying the control of transcription because the two phage have relatively small and therefore tractable genomes and yet exhibit a surprisingly complex set of effects upon one another. Each phage possesses the ability to drastically alter the expression of the other’s genome.

Expression of a late gene (R) of bacteriophage λ has been studied as a function of the state of five variables: the presence of the normal early rightwards promoter (pR), of an additional, immunity insensitive promoter (p′R), of immunity (im), of the N product (n) and of the Q product (q). Within the range of variables used, q appears as the necessary and sufficient condition for the expression of R (letting aside a minor component of late expression, which is regulated in an entirely distinct way: Dambly and Couturier, 1971).

The expression of many bacterial genes adapts itself in an almost instantaneous and reversible way to specific environmental changes. More specifically, the concentration of a number of metabolites, a function of the amounts of enzymes involved in their synthesis or degradation, in turn retroacts on the rate of synthesis of these enzymes. The genetic bases for this regulation were established by Jacob and Monod (1961). These authors also showed how the known elements of these regulatory mechanisms could be connected into a wide variety of circuits endowed with any desired degree of stability, in order to account for essentially irreversible processes like differentiation (Monod and Jacob, 1961). The general principles used by Jacob and Monod in their study of negative regulation were extended to positive regulation by Englesberg et al. (1965). An independent approach permitted the discovery of positive controls in temperate bacteriophages (see below, III).