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An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

ABSTRACT: The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

SUBMITTER: Jones TR

PROVIDER: S-EPMC321399 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

Jones Thomas R TR Lee Shi-Wu SW

Journal of virology 20040201 3

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures ...[more]

PMID: 14722304

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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:UnlabelledHuman cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence.ImportanceHuman cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and cellular protein-protein interactions to identify and understand the range of pUL103 functions. We found that pUL103 interacts with cellular antiviral defense systems and proteins involved in organelle biogenesis and transport of cytoplasmic vesicles and is present in infected cell nuclei. These results expand our understanding of the functional repertoire of pUL103 to include activities that extend from the earliest stages of infection through virion assembly and egress.

Dataset Information

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

Publications

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

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