scholarly journals The Cytoplasmic Tail of Glycoprotein M (gpUL100) Expresses Trafficking Signals Required for Human Cytomegalovirus Assembly and Replication

2007 ◽  
Vol 81 (19) ◽  
pp. 10316-10328 ◽  
Author(s):  
Magdalena Krzyzaniak ◽  
Michael Mach ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Cytoplasmic Tail ◽  
Viral Gene ◽  
Gene Encoding ◽  
Particle Assembly ◽  
Reading Frame ◽  
Virion Envelope

ABSTRACT The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.

Chapter 2a: Virology

2021 ◽  
Author(s):  
Daniel Růžek ◽  
Kentaro Yoshii ◽  
Marshall E. Bloom ◽  
Ernest A. Gould
Keyword(s):  
Host Cell ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Close Association ◽  
Spherical Particles ◽  
Structural Proteins ◽  
Membrane Structures ◽  
Vesicle Membrane ◽  
Reading Frame ◽  
Tbe Virus

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

scholarly journals Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

2006 ◽  
Vol 80 (11) ◽  
pp. 5611-5626 ◽  
Author(s):  
Jun-Young Seo ◽  
William J. Britt
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Tegument Protein ◽  
Reading Frame ◽  
Transfected Cells ◽  
Recombinant Viruses ◽  
Assembly Compartment ◽  
Sequence Requirements

ABSTRACT The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.

scholarly journals The Major Open Reading Frame of the β2.7 Transcript of Human Cytomegalovirus: In Vitro Expression of a Protein Posttranscriptionally Regulated by the 5′ Region

1998 ◽  
Vol 72 (10) ◽  
pp. 8425-8429 ◽  
Author(s):  
Giovanna Bergamini ◽  
Marko Reschke ◽  
Maria Concetta Battista ◽  
Maria Cristina Boccuni ◽  
Fabio Campanini ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Cytomegalovirus Infection ◽  
Intracellular Localization ◽  
Protein Product ◽  
Transient Transfection ◽  
Open Reading Frame ◽  
Major Open Reading Frame ◽  
Reading Frame ◽  
Human Cytomegalovirus Infection

ABSTRACT β2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5′ sequence of the transcript, which harbors two short upstream ORFs.

scholarly journals Posttranscriptional Suppression of Interleukin-6 Production by Human Cytomegalovirus

2005 ◽  
Vol 79 (1) ◽  
pp. 472-485 ◽  
Author(s):  
Claire Gealy ◽  
Marian Denson ◽  
Christine Humphreys ◽  
Brian McSharry ◽  
Gavin Wilkinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Interleukin 6 ◽  
Human Fibroblasts ◽  
Intracellular Localization ◽  
Viral Gene Expression ◽  
Suppressive Effect ◽  
Productive Infection ◽  
Viral Gene

ABSTRACT Human cytomegalovirus (HCMV) has evolved multiple strategies for suppression of the antiviral response of the infected cell. DNA array technology has revealed that HCMV clearly regulates host gene expression during the course of a productive infection by enhancing, sustaining, or suppressing steady-state levels of cellular transcripts. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in the immune response to infection. Here we report a detailed study of the effects of HCMV infection on IL-6 expression by human fibroblasts. UV-inactivated virus was found to induce high levels of IL-6 mRNA and protein expression, and IL-6 mRNA remained abundant in cells 16 h after inoculation even though the level of ongoing IL-6 transcription was not significantly enhanced. In lytic HCMV infections, the onset of viral gene expression resulted in two apparently antagonistic effects on IL-6 expression: (i) transcriptional activation, mediated at least in part by the IE2p86 protein, and (ii) posttranscriptional suppression mediated by destabilization of IL-6 mRNA. Transcriptional activation was outweighed by the suppressive effect, such that cells undergoing productive infection produced less IL-6 than cells challenged with inactivated virus. Suppression of IL-6 expression was independent of the viral IL-10 homologue, cmvIL-10. Destabilization of IL-6 mRNA was observed to coincide with the enhanced expression and aberrant intracellular localization of HuR, an mRNA-binding protein known to interact with IL-6 and other mRNAs containing 3′ AU-rich elements. Our data suggest a novel mechanism for gene regulation by HCMV at the posttranscriptional level.

scholarly journals Nuclear Localization of Tegument-Delivered pp71 in Human Cytomegalovirus-Infected Cells Is Facilitated by One or More Factors Present in Terminally Differentiated Fibroblasts

2010 ◽  
Vol 84 (19) ◽  
pp. 9853-9863 ◽  
Author(s):  
Rhiannon R. Penkert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Viral Gene Expression ◽  
Cell Types ◽  
Lytic Replication ◽  
Viral Gene ◽  
Differentiated Cells ◽  
Virion Envelope ◽  
Infected Cells

ABSTRACT Herpesviral virions contain a tegument layer that consists primarily of viral proteins. The delivery of fully functional proteins to infected cells upon virion envelope fusion to the plasma membrane allows herpesviruses to modulate cellular activities prior to viral gene expression. Certain tegument proteins can also regulate viral processes. For example, the pp71 tegument protein encoded by the UL82 gene of human cytomegalovirus (HCMV) stimulates viral immediate early (IE) gene expression and thus acts to initiate the productive lytic infectious cycle. In terminally differentiated fibroblasts infected with HCMV, tegument-delivered pp71 traffics to the nucleus and degrades the cellular transcriptional corepressor Daxx to initiate viral IE gene expression and lytic replication. However, when HCMV infects incompletely differentiated cells, tegument-delivered pp71 remains in the cytoplasm, allowing the nucleus-localized Daxx protein to silence viral IE gene expression and promote the establishment of a latent infection in certain cell types. We sought to determine whether undifferentiated cells block the trafficking of tegument-delivered pp71 to the nucleus or whether differentiated cells facilitate the nuclear transport of tegument-delivered pp71. Heterogenous cell fusion experiments demonstrated that tegument-delivered pp71 found in the cytoplasm of undifferentiated NT2 cells could be driven into the nucleus by one or more factors provided by fully differentiated fibroblasts. Our data raise the intriguing possibility that latency is the default program launched by HCMV upon viral entry into cells and that lytic infection is initiated only in certain (differentiated) cells that can facilitate the delivery of incoming pp71 to the nucleus.

scholarly journals Antisense Transcription in the Human Cytomegalovirus Transcriptome

2007 ◽  
Vol 81 (20) ◽  
pp. 11267-11281 ◽  
Author(s):  
Guojuan Zhang ◽  
Bindu Raghavan ◽  
Mark Kotur ◽  
Jacquelyn Cheatham ◽  
Daniel Sedmak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Antisense Transcription ◽  
Cdna Libraries ◽  
Viral Gene ◽  
Human Populations ◽  
Cdna Clones ◽  
Gene Products ◽  
Antisense Transcripts ◽  
Reading Frame ◽  
Viral Genes

ABSTRACT Human cytomegalovirus (HCMV) infections are prevalent in human populations and can cause serious diseases, especially in those with compromised or immature immune systems. The HCMV genome of 230 kb is among the largest of the herpesvirus genomes. Although the entire sequence of the laboratory-adapted AD169 strain of HCMV has been available for 18 years, the precise number of viral genes is still in question. We undertook an analysis of the HCMV transcriptome as an approach to enumerate and analyze the gene products of HCMV. Transcripts of HCMV-infected fibroblasts were isolated at different times after infection and used to generate cDNA libraries representing different temporal classes of viral genes. cDNA clones harboring viral sequences were selected and subjected to sequence analysis. Of the 604 clones analyzed, 45% were derived from genomic regions predicted to be noncoding. Additionally, at least 55% of the cDNA clones in this study were completely or partially antisense to known or predicted HCMV genes. The remarkable accumulation of antisense transcripts during infection suggests that currently available genomic maps based on open-reading-frame and other in silico analyses may drastically underestimate the true complexity of viral gene products. These findings also raise the possibility that aspects of both the HCMV life cycle and genome organization are influenced by antisense transcription. Correspondingly, virus-derived noncoding and antisense transcripts may shed light on HCMV pathogenesis and may represent a new class of targets for antiviral therapies.

scholarly journals Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

2010 ◽  
Vol 84 (7) ◽  
pp. 3162-3177 ◽  
Author(s):  
Sabarish V. Indran ◽  
Mary E. Ballestas ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Viral Assembly ◽  
Dominant Negative ◽  
Tegument Protein ◽  
Viral Glycoprotein ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.

Chapter 2a: Virology

2020 ◽  
Keyword(s):  
Host Cell ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Close Association ◽  
Spherical Particles ◽  
Structural Proteins ◽  
Membrane Structures ◽  
Vesicle Membrane ◽  
Reading Frame ◽  
Tbe Virus

TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). An additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalayan subtype (Himalayan and “178-79” group; TBEV-HIM). TBEV virions are small enveloped spherical particles about 50 nm in diameter. The TBEV genome consists of a single-stranded positive sense RNA molecule. The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

scholarly journals Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus

2004 ◽  
Vol 78 (1) ◽  
pp. 539-543 ◽  
Author(s):  
William J. Britt ◽  
Michael Jarvis ◽  
Jun-Young Seo ◽  
Derek Drummond ◽  
Jay Nelson
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Base Change ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Recombination System

ABSTRACT A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

Chapter 2a: Virology

2019 ◽  
Author(s):  
Daniel Růžek ◽  
Kentaro Yoshii ◽  
Marshall E. Bloom ◽  
Ernest A. Gould
Keyword(s):  
Host Cell ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Close Association ◽  
Spherical Particles ◽  
Structural Proteins ◽  
Membrane Structures ◽  
Vesicle Membrane ◽  
Reading Frame ◽  
Tbe Virus

• TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. • Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). Additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalaya subtype (Himalayan and “178-79” group; TBEV-HIM). • TBEV virions are small enveloped spherical particles about 50 nm in diameter. • The TBEV genome consists of a single-stranded positive sense RNA molecule. • The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). • The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. • The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). • TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.

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