Dataset Information

M94 is essential for the secondary envelopment of murine cytomegalovirus.

ABSTRACT: 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.

SUBMITTER: Maninger S

PROVIDER: S-EPMC3165754 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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M94 is essential for the secondary envelopment of murine cytomegalovirus.

Maninger Silke S Bosse Jens Bernhard JB Lemnitzer Frederic F Pogoda Madlen M Mohr Christian A CA von Einem Jens J Walther Paul P Koszinowski Ulrich H UH Ruzsics Zsolt Z

Journal of virology 20110629 18

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, ...[more]

PMID: 21715482

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Project description:Human cytomegalovirus (HCMV) secondary envelopment requires the viral tegument protein pUL71. The lack of pUL71 results in a complex ultrastructural phenotype with increased numbers of viral capsids undergoing envelopment at the cytoplasmic virus assembly complex. Here, we report a role of the pUL71 C terminus in secondary envelopment. Mutant viruses expressing C-terminally truncated pUL71 (TB71del327-361 and TB71del348-351) exhibited an impaired secondary envelopment in transmission electron microscopy (TEM) studies. Further mutational analyses of the C terminus revealed a tetralysine motif whose mutation (TB71mutK348-351A) resulted in an envelopment defect that was undistinguishable from the defect caused by truncation of the pUL71 C terminus. Interestingly, not all morphological alterations that define the ultrastructural phenotype of a TB71stop virus were found in cells infected with the C-terminally mutated viruses. This suggests that pUL71 provides additional functions that modulate HCMV morphogenesis and are harbored elsewhere in pUL71. This is also reflected by an intermediate growth defect of the C-terminally mutated viruses compared to the growth of the TB71stop virus. Electron tomography and three-dimensional visualization of different stages of secondary envelopment in TB71mutK348-351A-infected cells showed unambiguously the formation of a bud neck. Furthermore, we provide evidence for progressive tegument formation linked to advancing grades of capsid envelopment, suggesting that tegumentation and envelopment are intertwined processes. Altogether, we identified the importance of the pUL71 C terminus and, specifically, of a positively charged tetralysine motif for HCMV secondary envelopment.IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes severe symptoms, especially in immunocompromised hosts. Furthermore, congenital HCMV infection is the leading viral cause of severe birth defects. Development of antiviral drugs to prevent the production of infectious virus progeny is challenging due to a complex and multistep virion morphogenesis. The mechanism of secondary envelopment is still not fully understood; nevertheless, it represents a potential target for antiviral drugs. Our identification of the role of a positively charged motif in the pUL71 C terminus for efficient HCMV secondary envelopment underlines the importance of pUL71 and, especially, its C terminus for this process. It furthermore shows how cell-associated spread and virion release depend on secondary envelopment. Ultrastructural analyses of different stages of envelopment contribute to a better understanding of the mechanisms underlying the process of secondary envelopment. This may bring us closer to the development of novel concepts to treat HCMV infections.

Project description:The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXX?) in pUL71. This led us to hypothesize a requirement of the YXX? motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXX? motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXX? mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXX? motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-?-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC.IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to life-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins, such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and is regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy.

Project description:Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

Dataset Information

M94 is essential for the secondary envelopment of murine cytomegalovirus.

Publications

M94 is essential for the secondary envelopment of murine cytomegalovirus.

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