An immediate–late gene expression module decodes
            ERK
            signal duration

ABSTRACTViral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCECytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

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The Interaction between ORF18 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.01488-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 7

Author(s):

Angelica F. Castañeda ◽

Britt A. Glaunsinger

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Late Gene ◽

Central Component ◽

Gene Promoters ◽

Late Gene Expression ◽

Late Genes ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTIn the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral assembly and whose transcription initiates only after the onset of viral DNA replication. The vTAs in Kaposi’s sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins and how they coordinate to activate late gene promoters remain largely unknown. Here, we performed a comprehensive mutational analysis of the conserved residues in ORF18, which is a highly interconnected vTA component. Surprisingly, the mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical as even small disruptions in this complex result in profound defects in KSHV late gene expression.IMPORTANCEKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma and other B-cell cancers and remains a leading cause of death in immunocompromised individuals. A key step in the production of infectious virions is the transcription of viral late genes, which generates capsid and structural proteins and requires the coordination of six viral proteins that form a complex. The role of these proteins during transcription complex formation and the importance of protein-protein interactions are not well understood. Here, we focused on a central component of the complex, ORF18, and revealed that disruption of its interaction with even a single component of the complex (ORF30) prevents late gene expression and completion of the viral lifecycle. These findings underscore how individual interactions between the late gene transcription components are critical for both the stability and function of the complex.

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Roles of the Escherichia coli heat shock sigma factor 32 in early and late gene expression of bacteriophage T4.

Journal of Bacteriology ◽

10.1128/jb.170.3.1384-1388.1988 ◽

1988 ◽

Vol 170 (3) ◽

pp. 1384-1388 ◽

Cited By ~ 4

Author(s):

M W Frazier ◽

G Mosig

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Heat Shock ◽

Sigma Factor ◽

Bacteriophage T4 ◽

Late Gene ◽

Late Gene Expression

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Adenovirus Late-Phase Infection Is Controlled by a Novel L4 Promoter

Journal of Virology ◽

10.1128/jvi.00107-10 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7096-7104 ◽

Cited By ~ 22

Author(s):

Susan J. Morris ◽

Gillian E. Scott ◽

Keith N. Leppard

Keyword(s):

Gene Expression ◽

Intermediate Phase ◽

Expression Patterns ◽

Late Phase ◽

Adenovirus Vector ◽

Human Adenovirus ◽

Late Gene ◽

Genome Replication ◽

Late Gene Expression ◽

Viral Genome Replication

ABSTRACT During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

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