Dominant-Negative Proteins in Herpesviruses – From Assigning Gene Function to Intracellular Immunization

Mühlbach, Hermine; Mohr, Hermine; Ruzsics, Zsolt; Koszinowski, Ulrich H.

doi:10.3390/v1030420

Cited by 6 publications

(11 citation statements)

References 111 publications

(113 reference statements)

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“…This assay is tailored to identify inhibitory and dominant negative mutants (36). DN mutants are those inhibitory mutants that act within the pathway in which the wt protein is involved (16,29). With this assay we were able to find two N-terminal insertion mutants of pM94 which showed dominant negative activity.…”

Section: Discussionmentioning

confidence: 86%

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M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

Maninger

Bosse

Lemnitzer

et al. 2011

J Virol

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The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

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Section: Discussionmentioning

confidence: 86%

“…To achieve this, a DN protein must fulfill at least two functions. One function, which is usually based on biomolecular interactions, must remain intact, whereas another essential function must be disrupted by either deletion of a protein domain or its mutation, resulting in loss of function (29).…”

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confidence: 99%

M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

Maninger

Bosse

Lemnitzer

et al. 2011

J Virol

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“…In contrast to null mutants, which only reveal the dominant role of a protein, DN alleles have the potential to arrest viral pathways at different stages, thereby addressing multiple essential functions of a protein (reviewed in reference 39). Using in-depth functional knowledge and detailed experimental information regarding protein structure, such inhibitory mutants can be created by the targeted introduction of crucial, but subtle, mutations or by deleting a domain that represents an independent folding entity (18,39). However, such data are limited for most herpesvirus proteins.…”

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confidence: 99%

Characterization of Conserved Region 2-Deficient Mutants of the Cytomegalovirus Egress Protein pM53

Pogoda

Bosse

Wagner

et al. 2012

J Virol

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; therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.T he production and release of infectious herpesviral particles is a multistep process that begins in the nucleus of the infected cell, where viral genomes are packaged into nucleocapsids. A recent study of pseudorabies virus (PrV) showed that the nuclear envelope breaks down to allow the capsid to exit the nucleus (23). However, many studies have shown that the widely accepted nuclear egress pathway requires capsid translocation through the nuclear membrane via primary envelopment and de-envelopment. This process is facilitated by a number of viral and host cell protein interactions. From the viral side, the two major proteins involved are the gene products of UL31 and UL34 of HSV-1 (pUL31 and pUL34, respectively) or their homologues in all other herpesviruses studied to date, which promote efficient primary capsid envelopment at the inner nuclear membrane (INM) (11,24,40,50). The pUL31 homologue of murine cytomegalovirus (MCMV), pM53, is distributed throughout the nucleosol in the absence of other viral proteins. pM53 is targeted to the INM after interacting with the membrane protein pM50 (the homologue of pUL34) via a mechanism that is conserved throughout herpesvirus morphogenesis (13,27,31,46,55,56,62). pM50 and pM53 form the nuclear envelopment complex (NEC) and recruit other cellular and viral proteins, such as protein kinase C and pUS3 (3,8,22,37,40,41,47,53), resulting in displacement of the rigid nuclear lamina and nucleocapsid budding (reviewed in references 19 and 36).The mechanism by which the pUL31 and pUL34 homologues mediate capsid transition through the nuclear envelope is still not fully understood, but disruption of either partner usually leads to retention of viral capsids within the nucleus (5,11,13,24,31,50,64). Insights into this mechanism were gained from studying the PrV proteins pUL31 and pUL34, which induce vesicle formation in the nuclei of transfected cells in the absence of other viral components (20), supporting their role in the capsid envelopment process at the INM. The reciprocal binding sites, which result in NEC targeting to the INM, are well characterized in pM53 and pM50 and are conserved throughout the pUL31 family (5,13,14,30,31,54,56). Furthermore, recent studies of HSV-1 suggest a second essential interaction between the N-term...

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“…While the function of non-essential genes can be studied in the virus context using deletion and loss-of-function mutants, this approach is not applicable directly for essential genes. The study of null- or non-functional mutants of essential genes requires functional analysis by trans -complementation or the application of dominant negative (DN) mutants (reviewed in [11] , [12] ).…”

Section: Introductionmentioning

confidence: 99%

A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes

et al. 2014

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Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses.

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Dominant-Negative Proteins in Herpesviruses – From Assigning Gene Function to Intracellular Immunization

Cited by 6 publications

References 111 publications

M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

Characterization of Conserved Region 2-Deficient Mutants of the Cytomegalovirus Egress Protein pM53

A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes

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