Three-Dimensional Visualization of Gammaherpesvirus Life Cycle in Host Cells by Electron Tomography

Li, Peng; Ryazantsev, Sergey; Sun, Ren; Zhou, Z. Hong

doi:10.1016/j.str.2009.10.017

Cited by 63 publications

(58 citation statements)

References 51 publications

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“…The contacts of Nup358/RanBP2 with capsid-associated pUL36, possibly via importin ␤, may enable an initial docking of incoming HSV-1 capsids to the nuclear pores. Interactions between Nup214/CAN and capsid-associated pUL25 may then contribute to stabilizing the incoming capsids on the NPCs (25,26,62,64,84). An excess of pUL25 may have prevented an interaction of the incoming parental capsid-associated pUL25 with Nup214/ CAN by competitive inhibition.…”

Section: Discussionmentioning

confidence: 99%

“…17 in reference 82), those emerging from capsids of the gammaherpesvirus murine gammaherpesvirus 68 (MHV-68) docked at NPCs (see Fig. 3 in reference 64), and those emerging from HSV-1 capsids that had extruded their DNA in vitro (55,64). We therefore presume that these densities represent HSV-1 DNA genomes that emerged from incoming capsids and entered the nucleoplasm via the NPCs.…”

Section: ) or Mock-transfected Cells (Not Shown)mentioning

confidence: 98%

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Uncoupling Uncoating of Herpes Simplex Virus Genomes from Their Nuclear Import and Gene Expression

Rode

Döhner

Binz

et al. 2011

J Virol

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Incoming capsids of herpes simplex virus type 1 (HSV-1) enter the cytosol by fusion of the viral envelopes with host cell membranes and use microtubules and microtubule motors for transport to the nucleus. Upon docking to the nuclear pores, capsids release their genomes into the nucleoplasm. Progeny genomes are replicated in the nucleoplasm and subsequently packaged into newly assembled capsids. The minor capsid protein pUL25 of alphaherpesviruses is required for capsid stabilization after genome packaging and for nuclear targeting of incoming genomes. Here, we show that HSV-1 pUL25 bound to mature capsids within the nucleus and remained capsid associated during assembly and nuclear targeting. Furthermore, we tested potential interactions between parental pUL25 bound to incoming HSV-1 capsids and host factors by competing for such interactions with an experimental excess of cytosolic pUL25. Overexpression of pUL25, GFPUL25, or UL25GFP prior to infection reduced gene expression of HSV-1. Electron microscopy and in situ hybridization studies revealed that an excess of GFPUL25 or UL25GFP prevented efficient nuclear import and/or transcription of parental HSV-1 genomes, but not nuclear targeting of capsids or the uncoating of the incoming genomes at the nuclear pore. Thus, the uncoating of HSV-1 genomes could be uncoupled from their nuclear import and gene expression. Most likely, surplus pUL25 competed with important interactions between the parental capsids, and possibly between authentic capsid-associated pUL25, and cytosolic or nuclear host factors required for functional interaction of the incoming genomes with the nuclear machinery.Herpes simplex virus type I (HSV-1) is the most thoroughly studied pathogen among the alphaherpesviruses and among the eight human-pathogenic herpesviruses. Its double-stranded DNA genome of 152 kb is packaged into a capsid that is surrounded by 33 capsid-associated and tegument proteins, which in turn are wrapped by a viral envelope (43,51,68,73,93). HSV-1 assembly commences in the nucleus with the formation of spherical, scaffold-containing procapsids that develop into three types of angularized icosahedral capsids characterized by different sedimentation coefficients, morphologies, protein compositions, and mechanical properties (A, B, and C capsids) (3, 28, 75). The C capsids, which are the heaviest, enclose the viral genome; the B capsids still contain scaffold remnants but no DNA; and the A capsids are empty and lack both scaffold and DNA. After degradation of the scaffold proteins VP21 and VP22a by viral proteases, the procapsids package the viral DNA and mature into C capsids. Predominantly, C capsids leave the nucleus by primary envelopment and de-envelopment at nuclear membranes and acquire their final envelope by secondary envelopment in the cytoplasm (51,52,92,93). B and A capsids most likely result from a failure to initiate or to complete DNA packaging properly and are considered to be dead-end products that are formed during assembly (3, 85, 92).The major capsid prote...

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Section: Discussionmentioning

confidence: 99%

Section: ) or Mock-transfected Cells (Not Shown)mentioning

confidence: 98%

Uncoupling Uncoating of Herpes Simplex Virus Genomes from Their Nuclear Import and Gene Expression

Rode

Döhner

Binz

et al. 2011

J Virol

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“…Electron tomography tilt series were collected on an FEI Tecnai TF20 instrument with an accelerating voltage of 200 kV as previous described (Peng et al 2010). A Gatan cryoholder with a tilt range of À70 to +70 degrees was used and a 16-megapixel CCD camera was used to acquire the images.…”

Section: Tomography Data Acquisitionmentioning

confidence: 99%

DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

Faller¹,

Toso²,

Matsunaga³

et al. 2010

RNA

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DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.

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“…1B, in the nucleus, dispersed distribution of gold particles around intranuclear capsids (Fig. 1B, B1), as well as accumulation of gold particles in inclusion bodies formed during virion assembly and egress (29) (Fig. 1B, B2) and around nuclear membrane areas (Fig.…”

mentioning

confidence: 95%

Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68

Jia

Shen

et al. 2016

J Virol

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cTegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses; however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus 68 (MHV-68) ORF45 and found that this protein was incorporated into virions around the site of host-derived vesicles. Notably, the absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.A s a unique structure of herpesviruses, tegument functions in multiple aspects of the viral life cycle, including modulation of the host cellular environment and initiation of viral gene transcription at the immediate-early phase of virus infection, transport of nucleocapsids to the nuclear pore, and virion morphogenesis and egress (1-10). Virion morphogenesis is a complicated event that takes place in the late phase of the herpesvirus lytic life cycle. After acquisition of viral genomes in the nucleus, nucleocapsids bud at the inner nuclear membrane to form primary virions in the perinuclear space, followed by deenvelopment at the outer nuclear membrane, releasing capsids into the cytoplasm. Capsids then undergo tegumentation and reenvelopment to obtain tegument proteins and envelope glycoproteins (11,12,13). Finally, mature virions are released from the cell.ORF45 is a virion-associated protein that is conserved in gammaherpesviruses but has no homologue in alpha-or betaherpesviruses. The ORF45 homologues in Kaposi's sarcoma-associated herpesvirus (KSHV), murine gammaherpesvirus 68 (MHV-68), Epstein-Barr virus (EBV), and rhesus rhadinovirus (RRV) are 407 amino acids (aa), 206 aa, 217 aa, and 353 aa, respectively (14, 15). The homology mainly lies in the C-terminal region of the protein. Among these homologues, KSHV ORF45 is the best studied. It is virion associated (16,17) and is a multifunctional protein. It interacts with interferon regulatory factor 7 and inhibits virus-induced interferon production, leading to viral immune evasion (2, 18). Characterization of a KSHV ORF45-null bacterial artificial chromosome (BAC) showed that although ORF45 is not essential for KSHV lytic replication, it plays roles in both early and late stages of viral infection (19). Indeed, ORF45 was shown to interact with kinesin-2 to mediate the transport of viral particles along microtubules (4). ORF45 also interacts with another tegument protein, ORF33, and plays a crucial role in efficient production of KSHV viral particles (15). Most recently, ORF45 was shown to mediate association of KSHV particles with internal lipid rafts for viral assembly and egress (20).MHV-68 ORF45 has also been identified as a virion-associated protein (21,22,23). Biochemical analysis demonstrated that ORF45 is a true tegument protein that is packaged into virions (23). By construction and analysis of an ORF45-null MHV-68 mutant using a BAC system, Jia et al. demonstrated that the ORF45-null MH...

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Three-Dimensional Visualization of Gammaherpesvirus Life Cycle in Host Cells by Electron Tomography

Cited by 63 publications

References 51 publications

Uncoupling Uncoating of Herpes Simplex Virus Genomes from Their Nuclear Import and Gene Expression

Uncoupling Uncoating of Herpes Simplex Virus Genomes from Their Nuclear Import and Gene Expression

DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68

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