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15.3.1.1.1: Herpesviruses - Biology

15.3.1.1.1: Herpesviruses - Biology


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Herpes viruses cause a wide range of latent, recurring infections including oral and genital herpes, cytomegalovirus, and chicken pox.

Learning Objectives

  • Recognize the attributes of herpes viruses

Key Points

  • Herpesviridae is a large family of DNA viruses that cause diseases in animals, including humans.
  • The structure of herpes viruses consists of a relatively large double-stranded, linear DNA genome encased within an icosahedral protein cage called the capsid, which is wrapped in a lipid bilayer called the envelope.
  • Notable herpes viruses include herpes simplex viruses 1 and 2, Varicella zoster virus (the causative agent of shingles and chicken pox), cytomegalovirus, and Kaposi’s sarcoma virus.
  • There is no method to eradicate herpes virus from the body, but antiviral medications, such as acyclovir, can reduce the frequency, duration, and severity of outbreaks.

Key Terms

  • tegument: A natural covering of the body or of a bodily organ.
  • capsid: The outer protein shell of a virus.
  • virion: A single individual particle of a virus (the viral equivalent of a cell).

Herpesviridae is a large family of DNA viruses that cause diseases in animals, including humans. The members of this family are also known as herpes viruses. The family name is derived from the Greek word herpein (“to creep”), referring to the latent, recurring infections typical of this group of viruses.

Animal herpes viruses all share some common properties. The structure of these viruses consists of a relatively large double-stranded, linear DNA genome encased within an icosahedral protein cage called the capsid, which is wrapped in a lipid bilayer called the envelope. The envelope is joined to the capsid by means of a tegument. This complete particle is known as the virion. HSV-1 and HSV-2 each contain at least 74 genes within their genomes, although speculation over gene crowding allows as many as 84 unique protein-coding genes by 94 putative pen reading frames. These genes encode a variety of proteins involved in forming the capsid, tegument and envelope of the virus, as well as controlling the replication and infectivity of the virus.

Types of herpes viruses

There are nine distinct herpes viruses which cause disease in humans:

  • HHV‑1 Herpes simplex virus-1 (HSV-1)
  • HHV-2 Herpes simplex virus-2 (HSV-2)
  • HHV-3 Varicella zoster virus (VZV)
  • HHV-4 Epstein-Barr virus (EBV)
  • HHV-5 Cytomegalovirus (CMV)
  • HHV-6A/B Roseolovirus, Herpes lymphotropic virus
  • HHV-7 Pityriasis Rosea
  • HHV-8 Kaposi’s sarcoma-associated herpesvirus

Of particular interest include HSV-1 and HSV-2, which cause oral and/or genital herpes, HSV-3 which causes chickenpox and shingles, and HHV-5 which causes mononucleosis-like symptoms, and HHV-8 which causes a Kaposi’s sarcoma, a cancer of the lymphatic epithelium.

Infection is caused through close contact with an infected individual. Infection is initiated when a viral particle comes in contact with the target cell specific to the individual herpes virus. Viral glycoproteins bind cell surface receptors molecules on the cell surface, followed by virion internalization and disassembly. Viral DNA then migrates to the cell nucleus where replication of viral DNA and transcription of viral genes occurs.

During symptomatic infection, infected cells transcribe lytic viral genes. In some host cells, a small number of viral genes termed latency-associated transcripts accumulate instead. In this fashion, the virus can persist in the cell (and thus the host) indefinitely. While primary infection is often accompanied by a self-limited period of clinical illness, long-term latency is symptom-free.

Reactivation of latent viruses

This has been implicated in a number of diseases (e.g. Shingles, Pityriasis Rosea). Following activation, transcription of viral genes transitions from latency-associated transcripts to multiple lytic genes; these lead to enhanced replication and virus production. Often, lytic activation leads to cell death. Clinically, lytic activation is often accompanied by emergence of non-specific symptoms such as low grade fever, headache, sore throat, malaise, and rash, as well as clinical signs such as swollen or tender lymph nodes, and immunological findings such as reduced levels of natural killer cells.

There is no method to eradicate the herpes virus from the body, but antiviral medications, such as acyclovir, can reduce the frequency, duration, and severity of outbreaks. Analgesics such as ibuprofen and acetaminophen can reduce pain and fever. Topical anesthetic treatments such as prilocaine, lidocaine, benzocaine or tetracaine can also relieve itching and pain.


15.3.1.1.1: Herpesviruses - Biology

  • Core. The core consists of a single linear molecule of dsDNA in the form of a torus.
  • Capsid. Surrounding the core is an icosahedral capsid with a 100 nm diameter constructed of 162 capsomeres.
  • Tegument. Between the capsid and envelope is an amorphous, sometimes asymmetrical, feature named the tegument. It consists of viral enzymes, some of which are needed to take control of the cell's chemical processes and subvert them to virion production, some of which defend against the host cell's immediate responses, and others for which the function is not yet understood.
  • Envelope. The envelope is the outer layer of the virion and is composed of altered host membrane and a dozen unique viral glycoproteins. They appear in electron micrographs as short spikes embedded in the envelope.

The genes are characterized as either essential or dispensable for growth in cell culture. Essential genes regulate transcription and are needed to construct the virion. Dispensable genes for the most part function to enhance the cellular environment for virus production, to defend the virus from the host immune system and to promote cell to cell spread. The large numbers of dispensable genes are in reality required for a productive in vivo infection. It is only in the restricted environment of laboratory cell cultures that they are dispensable.

All herpesvirus genomes contain lengthy terminal repeats both direct and inverted. There are six terminal repeat arrangements and understanding how these repeats function in viral success is an interesting part of current research.


FAQs About EEHV

What do we know about elephant herpesviruses?

To date, scientists have identified 14 genetically different elephant herpesviruses (EEHV), most of which are known to cause hemorrhagic disease. The viruses found in symptomatic elephants at different zoos and other institutions are genetically distinct, which means that they are not all the same strain spread by the transfers of elephants between and among zoos. The most common cause of acute EEHV cases and deaths in elephants is the EEHV 1A strain.

Herpesviruses are widespread in all vertebrate taxa, including humans. While herpesviruses are usually species specific, they can affect closely related species. (EEHV does not pose a health risk to humans, though humans are host to their own strains of herpesviruses). All herpesviruses do share common some features. Once inside a host, the virus can go into a latent (hidden) phase after causing only mild symptoms or no signs of disease at all. Scientists do not yet know where in the body EEHV resides during the latent phase.

For reasons unknown, an elephant herpesvirus can come out of latency and circulate throughout the bloodstream, causing disease. This is the only time when a herpesvirus can be readily detected in blood samples. Reliable tests are not yet available to detect a latent infection. Most elephants are able to fight the virus and survive when it comes out of latency. Calves appear to be most susceptible to EEHV disease after they have been weaned, at a time when they are not protected by their mother’s antibodies.


HVint: A Strategy for Identifying Novel Protein-Protein Interactions in Herpes Simplex Virus Type 1

Human herpesviruses are widespread human pathogens with a remarkable impact on worldwide public health. Despite intense decades of research, the molecular details in many aspects of their function remain to be fully characterized. To unravel the details of how these viruses operate, a thorough understanding of the relationships between the involved components is key. Here, we present HVint, a novel protein-protein intraviral interaction resource for herpes simplex virus type 1 (HSV-1) integrating data from five external sources. To assess each interaction, we used a scoring scheme that takes into consideration aspects such as the type of detection method and the number of lines of evidence. The coverage of the initial interactome was further increased using evolutionary information, by importing interactions reported for other human herpesviruses. These latter interactions constitute, therefore, computational predictions for potential novel interactions in HSV-1. An independent experimental analysis was performed to confirm a subset of our predicted interactions. This subset covers proteins that contribute to nuclear egress and primary envelopment events, including VP26, pUL31, pUL40, and the recently characterized pUL32 and pUL21. Our findings support a coordinated crosstalk between VP26 and proteins such as pUL31, pUS9, and the CSVC complex, contributing to the development of a model describing the nuclear egress and primary envelopment pathways of newly synthesized HSV-1 capsids. The results are also consistent with recent findings on the involvement of pUL32 in capsid maturation and early tegumentation events. Further, they open the door to new hypotheses on virus-specific regulators of pUS9-dependent transport. To make this repository of interactions readily accessible for the scientific community, we also developed a user-friendly and interactive web interface. Our approach demonstrates the power of computational predictions to assist in the design of targeted experiments for the discovery of novel protein-protein interactions.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.


SV40 and Polyomavirus

The best studied DNA tumor viruses, from the standpoint of molecular biology, are probably simian virus 40 (SV40) and polyomavirus. Although neither of these viruses is associated with human cancer, they have been critically important as models for understanding the molecular basis of cell transformation. The utility of these viruses in cancer research has stemmed from the availability of good cell culture assays for both virus replication and transformation, as well as from the small size of their genomes (approximately 5 kb).

SV40 and polyomavirus do not induce tumors or transform cells of their natural host species—monkeys and mice, respectively. In cells of their natural hosts (permissive cells), infection leads to virus replication, cell lysis, and release of progeny virus particles (Figure 15.13). Since a permissive cell is killed as a consequence of virus replication, it cannot become transformed. The transforming potential of these viruses is revealed, however, by infection of nonpermissive cells, in which virus replication is blocked. In this case, the viral genome sometimes integrates into cellular DNA, and expression of specific viral genes results in transformation of the infected cell.

Figure 15.13

SV40 replication and transformation. Infection of a permissive cell results in virus replication, cell lysis, and release of progeny virus particles. In a nonpermissive cell, virus replication is blocked, allowing some cells to become permanently transformed. (more. )

The SV40 and polyomavirus genes that lead to cell transformation have been identified by detailed molecular analyses. The viral genomes and mRNAs have been completely sequenced, viral mutants that are unable to induce transformation have been isolated, and the transforming potentials of individual viral genes have been determined by gene transfer assays. Transformation by these viruses has thus been found to result from expression of the same viral genes that function in early stages of lytic infection. The genomes of SV40 and polyomavirus are divided into early and late regions. The early region is expressed immediately after infection and is required for synthesis of viral DNA. The late region is not expressed until after viral DNA replication has begun, and includes genes encoding structural components of the virus particle. The early region of SV40 encodes two proteins, called small and large T antigens, of about 17 kd and 94 kd, respectively (Figure 15.14). Their mRNAs are generated by alternative splicing of a single early-region primary transcript. Polyomavirus likewise encodes small and large T antigens, as well as a third early-region protein of about 55 kd, designated middle T. Transfection of cells with cDNAs for individual early-region proteins has shown that SV40 large T is sufficient to induce transformation, whereas middle T is primarily responsible for transformation by polyomavirus.

Figure 15.14

The SV40 genome. The genome is divided into early and late regions. Large and small T antigens are produced by alternative splicing of early-region pre-mRNA.

During lytic infection, these early-region proteins fulfill multiple functions required for virus replication. SV40 T antigen, for example, binds to the SV40 origin and initiates viral DNA replication (see Chapter 5). In addition, the early-region proteins of SV40 and polyomavirus stimulate host cell gene expression and DNA synthesis. Since virus replication is dependent on host cell enzymes (e.g., DNA polymerase), such stimulation of the host cell is a critical event in the viral life cycle. Most cells in an animal are nonproliferating, and therefore must be stimulated to divide in order to induce the enzymes needed for viral DNA replication. This stimulation of cell proliferation by the early gene products can lead to transformation if the viral DNA becomes stably integrated and expressed in a nonpermissive cell.

As discussed later in this chapter, both SV40 and polyomavirus early-region proteins induce transformation by interacting with host proteins that regulate cell proliferation. For example, SV40 T antigen binds to and inactivates the host cell tumor suppressor proteins Rb and p53, which are key regulators of cell proliferation and cell cycle progression (see Figures 14.20 and 14.21).


Retrieval of sequences

All virus miRNA sequences (mature and precursor miRNAs) were obtained from the microRNA database miRBase (v22.1) [34]. Full-length herpesvirus genomes were obtained from NCBI GenBank and ViPR database (http://www.viprbrc.org) [70].

The upstream sequences of precursor miRNAs of all virus strains under study were obtained as follows. First, if a precursor miRNA overlaps with another gene and were unidirectional, the 1000 bp region upstream of the concerned gene was obtained on the other hand, if a precursor miRNA and the gene were convergent, the 1000 bp region upstream of the precursor miRNA was obtained. Second, if precursor microRNAs were known to be intergenic, the 1000 bp region upstream of the precursor miRNA was retrieved.

PQS mapping

The retrieved upstream sequences were analyzed using Quadparser (a computer algorithm) [61] to identify PQS with parameters (minimum G-tetrad-3 and loop length- 1-15). PQS density was defined as the total number of non-overlapping PQS predicted per kilo base of the sequence analyzed. Average PQS densities were computed for analysis.

Randomization of sequences

In order to determine whether the occurrence of PQS motifs in the retrieved sequences is a random/non-random event, the selected sequences were shuffled while preserving the dinucleotide frequencies. This was achieved by performing a dinucleotide shuffle of the selected sequences (without changing the overall nucleotide composition). To do so, the base pairs were selected by randomly generated base numbers and the Eulerian walk method was employed while satisfying the constraint of keeping the number of dinucleotides constant before and after shuffling. The shuffling were performed 5 times. The python script (Additional file 2) used for dinucleotide shuffling of the 1000 bp sequences under study, is based on the freely available ‘uShuffle’ program script [71] with some modifications to facilitate easy analysis of the necessary parameters. Additional details are provided in a ‘Readme’ text file. Average PQS densities were mapped in the randomized sequences generated and were compared to that in the native sequences.

PQS conservation analysis

Upstream 1 kb sequences of herpesvirus miRNA promoters possessing at least 1 PQS motif (identified in the full-length virus sequences) were retrieved and studied for conservation analysis by performing multiple sequence alignment. Sequences for full length virus strains were downloaded from NCBI GenBank and ViPR database (http://www.viprbrc.org) [70]. Accession numbers of all sequences analyzed are mentioned in Additional file 1 Table S4. PQS motifs with intact Gs in the consecutive G-tetrads were considered conserved. Loop sequences with variable length and composition were not taken into account for conservation analysis.

Circular Dichroism spectroscopy and melting studies

CD studies were performed on a Chirascan circular dichroism spectrometer (Applied Photophysics Limited, UK). The sequences of the 2 PQS-motifs used (wild type and mutant) are listed in Additional file 1 Table S2. The oligonucleotides were purchased from Integrated DNA Technologies (IDT) for all biophysical experiments. Oligonucleotides (10 μM) were dissolved in 10 mM sodium cacodylate buffer (pH -7.5) along with 100 mM potassium chloride (KCl). The samples were heated at 95 °C for 5 min and slowly cooled to room temperature. A quartz cuvette (1 mm path length) was used for recording of spectra at the wavelength range (220–320 nm) with a 1 nm bandwidth, 1 nm step size and time of 1 s per point at 20 °C. CD melting was performed at a fixed concentration of oligonucleotides (10 μM), either with or without a fixed concentration of G-quadruplex ligands TMPyP4 and pyridostatin (PDS). The data was recorded at a ramp rate of 1 °C/minute over a range of 20–93 °C. A buffer baseline was recorded and subtracted from the sample spectra. Tm (melting temperature) was calculated by the first derivative method. Final analysis of the data was conducted using Origin 9.1 (Origin Lab Corp.).

NMR spectroscopy

The oligonucleotide samples were heated at 95 °C for 5 min and slowly cooled to room temperature. The NMR sample contained 300 μM oligonucleotides in 20 mM potassium phosphate buffer (pH 7.0), 100 mM KCl and 10% D2O (v/v). 1D 1 H NMR spectra were recorded using Bruker Avance III spectrometer equipped with cryogenic 5 mm TCI triple-resonance probe, operating at a field strength of 500 MHz. The spectra were recorded at 20 °C using Topspin 3.5 (Bruker AG). Data processing and analysis were performed with Topspin 4.6 software (Bruker AG).

Polyacrylamide gel electrophoresis

Oligonucleotides were prepared at 10 μM concentration in Tris-EDTA buffer (pH -7.0) and 100 mM KCl. The samples were heated at 95 °C for 5 min and slowly cooled to room temperature before loading. Native and denaturing polyacrylamide gels were prepared in 1× Tris-borate EDTA (TBE) buffer. 7 M urea was used as a denaturant to prepare denaturing polyacrylamide gel. Gels were run in 0.5× TBE with 50 mM KCl.

UV melting studies

A Cary 100 Bio UV-Vis double-beam spectrophotometer (Agilent Technologies) equipped with a multi-cell holder attached to a Peltier controller was used to perform UV melting experiments. Oligonucleotides at a concentration of 4 μM were mixed with 10 mM sodium cacodylate (pH -7.5) and 100 mM KCl. For ligand studies, fixed concentrations of TMPyP4 and PDS were used. The melting curves were recorded at 295 nm both ways (melting and annealing) between 20 °C and 95 °C with a ramp rate of 1 °C/min. Origin 9.1 (Origin Lab Corp.) was used to analyze and plot melting curves.

Luciferase constructs

The native promoter of KSHV miR-K12 cluster was amplified by PCR from KSHV JSC-1 genomic DNA which was kindly provided by Dr. Tathagata Choudhuri (Visva Bharati University, West Bengal, India), while HCMV miR-US33 promoter region was commercially synthesized by Life Technologies Corp. The wild type and mutant promoters were cloned in pGL3-basic vector (Promega) upstream of firefly luciferase coding sequence using appropriate primers listed in Additional file 1 Table S3. The plasmid constructs were extracted using QIAprep Spin Miniprep Kit (Qiagen) and confirmed by sequencing.

Cell proliferation assay (MTT)

Cell proliferation assay was performed in 96 well plate by incubating HEK293T cells (seeding density = 1 × 10 4 cells/well) in the presence of multiple doses of TMPyP4 (Sigma) or Pyridostatin (Sigma). After 24 h, cells were exposed to MTT (3-(4,5-Dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) reagent for 1 h. The medium was replaced with 100 μl dimethylsulfoxide (DMSO) and optical density measured at 570 nm (Additional file 1, Figure S1).

Luciferase reporter assay

HEK293T cells (procured from NCCS, Pune, India) were maintained in Dulbecco’s modified medium (Invitrogen) supplemented with 10% fetal bovine serum and were incubated at 37 °C and with 5% CO2. HEK293T cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well 24 h prior to transfection. The luciferase reporter constructs (wild-type or mutant 500 ng each) and 20 ng of pRL-TK (25:1 ratio) were co-transfected using PEI (polyethylenimine) into HEK293T cells in 24-well plates. For ligand studies, G-quadruplex ligands TMPyP4 and Pyridostatin were added 2 h after transfection at the appropriate concentration. Both ligands were used in the absence of light. At 24 h post-transfection, cell lysates were prepared using passive lysis buffer. Luciferase assays were performed using a dual luciferase reporter assay system according to the manufacturer’s protocols (Promega) with MicroBeta2 Microplate Scintillation Counter (Perkin Elmer). Firefly luciferase activity was normalized to renilla luciferase activity. Three independent experiments were done in triplicates.

Data analyses

Data was plotted as mean values ± SD in at least three distinct experiments. The statistically significant difference was defined as P < 0.01 calculated using Student’s t-test unless mentioned otherwise. Figure 1 and Fig. 3 were made using Microsoft Powerpoint. R software was used to generate violin plot (Fig. 2a). Origin 9.1 (Origin Lab Corp.) was used to plot melt curves and bar graphs.


Herpes Virus Replication

Department of Biochemistry and Molecular Biology University of Miami School of Medicine P.O. Box 016129 Miami Florida 33101-6129 USA.

Department of Biochemistry and Molecular Biology University of Miami School of Medicine P.O. Box 016129 Miami Florida 33101-6129 USA.

Department of Biochemistry and Molecular Biology University of Miami School of Medicine P.O. Box 016129 Miami Florida 33101-6129 USA.

Department of Biochemistry and Molecular Biology University of Miami School of Medicine P.O. Box 016129 Miami Florida 33101-6129 USA.

Abstract

Herpesviruses are large double stranded DNA animal viruses with the distinguishing ability to establish latent, life-long infections. To date, eight human herpesviruses that exhibit distinct biological and corresponding pathological/clinical properties have been identified. During their life cycles, herpesviruses execute an intricate chain of events geared towards optimizing their replication. This sets an interesting paradigm to study fundamental biological processes. This review summarizes recent developments in herpesvirus research with emphasis on genome transactions, particularly with respect to the prototypic herpes simplex virus type-1. IUBMB Life, 55: 13-22, 2003


15.3.1.1.1: Herpesviruses - Biology

The alpha herpesviruses are an important group of viruses characterized by a short reproductive cycle, rapid destruction of the host cell, and the ability to replicate in a wide variety of host tissues. A key attribute of these viruses is the ability to establish lifelong latent infection in the peripheral nervous system of the natural host. Research into the molecular and cellular biology of the alpha herpesviruses has advanced greatly in recent years.

Written by internationally recognized experts, this book highlights the more provocative and exciting findings in herpesvirus research. Each chapter is a review of a specific area with an emphasis on recent advances and the latest developments. Topics include: multifunctional proteins, advances in DNA replication, new information on the regulation of gene expression, the emergence of new technologies, recent technological advances in fluorescent probes, the induction of apoptosis, the disruption of interferon, vaccine development and drug design.

With a specific focus on new and topical herpesvirus research, this book is essential reading for everyone with an interest in herpesviruses and recommended reading for other scientists working in viral pathogenesis, viral genomics and antiviral research.

"a very good book" from Antiviral Chemistry and Chemotherapy 17: 355 (2006)

"This book presents a series of well written chapters on aspects of human alpha herpesvirus biology and will be a useful library resource" from Microbiology Today (2007)

(EAN: 9781904455097 9781913652289 Subjects: [virology] [microbiology] [medical microbiology] [molecular microbiology] )


Introduction

Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) are important human pathogens. It is estimated that approximately 90% of the world’s population are infected with one or both viruses [1]. HSV-1 is the primary cause of cold sores and HSV-2 of genital herpes. These conditions are both highly contagious, and HSV-2 is amongst the most common sexually transmitted infections. Infection with HSV is lifelong, owing to the ability of herpesviruses to enter a latent state with periodic reactivations [2]. HSV can also cause more serious conditions including keratitis, which may lead to loss of sight [3], and a potentially fatal encephalitis [4]. The herpesvirus family includes many other important human pathogens, such as varicella-zoster virus, the cause of chicken pox and shingles cytomegalovirus, a notable cause of congenital abnormalities Kaposi sarcoma–associated herpesvirus, which causes cancer in immune-compromised individuals and Epstein–Barr virus, the cause of infectious mononucleosis that has also been linked to several cancers.

Herpesviruses are large double-stranded DNA viruses, having genomes up to 240 kbp. The viral DNA is packaged in a complex T = 16 icosahedral capsid that is 1,250 Å in diameter [5,6]. The DNA-containing capsid, or nucleocapsid, is embedded in a proteinaceous layer known as the tegument that is in turn surrounded by a host-derived lipid envelope. The viral envelope is studded with glycoproteins that mediate viral attachment and entry. HSV virions enter host cells by fusing their envelopes with the host cell plasma membrane, allowing the nucleocapsid and tegument to enter the cytoplasm [7]. The nucleocapsid traffics along microtubules to the microtubule-organising centre, and from there, to the nucleus [8]. The nucleocapsid then docks to a nuclear pore complex, through which it injects its genome into the nucleus [9,10]. DNA egress from the capsid is through a unique portal-vertex, located at an icosahedral 5-fold symmetry axis.

The portal-vertex is also the means by which the viral DNA is packaged into capsids within the nucleus [11]. Virion morphogenesis commences in the nucleus with the formation of the procapsid an icosahedrally symmetrical spherical shell assembly of capsomeres that are hexamers (hexons) and pentamers (pentons) of the major capsid protein pUL19 (VP5) [12]. Heterotrimers of pUL38 (VP19C) and pUL18 (VP23)—termed triplexes—along with the scaffold protein pUL26, direct procapsid assembly, which nucleates around the dodecameric portal formed by pUL6 [13]. Procapsid maturation—angularisation and expulsion of the scaffold protein—occurs as the viral genome is pumped into the shell [14]. Replication of the viral genome results in formation of a concatemer, from which unit-length genomes are packaged into procapsids by the portal (pUL6) and terminase complex consisting of pUL33, pUL28, and pUL15 [15,16]. It has been noted that herpesviruses are structurally and biologically similar to DNA-containing tailed bacteriophages, and it has been suggested that these two viral groups share a common ancestry. This is based on the observation of fold conservation in the major capsid protein [17] and their similar capsid assembly and DNA-packaging strategies. Sequence analysis also indicates that pUL15 is a homolog of the phage terminase large subunit. By analogy, pUL15 is predicted to have ATPase activity, powering the translocation of the genome into the capsid through the portal, and endonuclease activity cleaving the DNA when a cleavage signal is detected [18]. pUL28 is known to bind viral DNA and is equivalent to the small terminase subunit of bacteriophages [19].

The capsid-associated tegument complex (CATC—previously termed CCSC and CVSC), is composed of pUL17, pUL25, and pUL36 and binds to the triplexes and hexons about the icosahedral 5-fold vertices of mature capsids within the nucleus [5,20–25]. Notably, it has been observed that pUL25 is essential for retention of DNA within the nucleocapsid [26,27]. Moreover, pUL25 has also been shown to be important for genome release [28].

The mature nucleocapsid leaves the nucleus by budding through the nuclear membrane via an envelopment/de-envelopment step [29]. Cytoplasmic capsids acquire further tegument proteins in the cytoplasm and are enveloped by budding into plasma membrane–derived lipid vesicles, from which they are released by exocytosis at the cell surface [30].

The structure of the HSV particle has been the subject of investigation for over 30 years [31], using cryoEM and icosahedral 3D reconstruction to determine the high-resolution features of the nucleocapsid [5,25,32,33] and lower-resolution tomography to investigate the nature of asymmetric features such as the portal-vertex and viral envelope [34,35]. Attempts to resolve the structure of the portal-vertex have been largely unsuccessful, however. This is because the herpesvirus portal-vertex is similar in size and mass to the penton-vertex, unlike bacteriophages, in which the portal-vertex is marked by the presence of a substantial tail assembly. Moreover, in herpes virions, the subtle differences between the portal-vertex and penton-vertices are obscured by the tegument layer. Finally, the high symmetry of the viral capsid dominates attempts to align particle images for asymmetric reconstruction.

Here, we show the structure of the portal-vertex of HSV-1 at 8 angstroms resolution, revealed by focussed-classification [36,37] and 3D reconstruction of cryoEM images of purified virions. These data reveal that the usual pUL19 penton is replaced by a unique 5-fold symmetrical assembly. This feature displays five well-defined coiled-coil motifs, each made up of two α-helices, arranged perpendicular to the capsid surface about the 5-fold symmetry axis. It appears to be anchored to the virion by interactions with triplex-like structures that occupy the position normally taken up by peripentonal Ta triplexes, immediately about the 5-fold vertex. The CATC assembly is still present and, similarly to penton associated CATC, is bound to the Tc triplexes, forming a bridge across the periportal triplex-like structures towards the 5-fold axis. We interpret our data as showing that the pUL25 C-terminal domains are positioned differently to those seen at penton-vertices, giving rise to a small tail-like assembly that crowns the unique 5-fold vertex. Strong density was seen to extend through the portal-vertex structures that we interpret as DNA. This suggests that the trailing end of the packaged genome remains engaged in the portal-vertex, ready for release through the nuclear pore. The portal itself is not well resolved owing to a mismatch between the C5 symmetry imposed in calculating our reconstruction and the C12 symmetry of that feature. Finally, our reconstruction also reveals the arrangement of packaged DNA within the virion, which is clearly resolved as a left-handed spool arranged in concentric layers.

We provide the highest-resolution view to date of a critical component of the herpesvirus virion. The portal-vertex is a molecular machine responsible for both packaging and release of the viral genome, in one of the most important groups of viral pathogens to infect humans. Furthermore, our focussed classification approach demonstrates the power of modern image-processing algorithms to break the shackles of symmetry that have limited our understanding of virus structural biology for so long.


Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging

The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids-which develop into infectious virions-are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled.

Importance: In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation damage was used to localize internal proteins of HSV-1, yielding insights into how capsid maturation is regulated. The scaffolding protein, which forms inner shells in the procapsid and B capsid, is exceptionally bubbling-prone. In the mature DNA-filled C capsid, a previously undetected protein was found to underlie the icosahedral vertices: this is tentatively assigned as a storage form of the viral protease. We also observed a capsid species that appears to contain substantial amounts of scaffolding protein as well as DNA, suggesting that DNA packaging and expulsion of the scaffolding protein are coupled processes.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

Schematic diagram of the molecular…

Schematic diagram of the molecular anatomy of four HSV-1 capsids. UL19, the major…

(A) Field of procapsids in a low-dose image (first exposure) (B) incipient bubbling…

Bubbling of the procapsid scaffolding…

Bubbling of the procapsid scaffolding shell and surface shell at high doses. In…

A dose series of cryo-electron…

A dose series of cryo-electron micrographs of a field containing B capsids, R…

A dose series of cryo-electron…

A dose series of cryo-electron micrographs of a mixed field of A capsids,…

Central sections of 3D reconstructions…

Central sections of 3D reconstructions from a dose series of C capsids. For…

(A) Distinguishing vertex-associated bubbles from…

(A) Distinguishing vertex-associated bubbles from other bubbles in C capsids, illustrated for three…

Cryo-micrographs and bubblegrams of two…

Cryo-micrographs and bubblegrams of two hyperbubblers compared with A capsids, B capsids, and…

Model of a portion of the HSV-1 procapsid surface viewed from inside and…


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The Baltimore classification system is used to group viruses together based on their manner of messenger RNA (mRNA) synthesis and is often used alongside standard virus taxonomy, which is based on evolutionary history. DNA viruses constitute two Baltimore groups: Group I: double-stranded DNA viruses, and Group II: single-stranded DNA viruses. While Baltimore classification is chiefly based on transcription of mRNA, viruses in each Baltimore group also typically share their manner of replication. Viruses in a Baltimore group do not necessarily share genetic relation or morphology. [1]

Double-stranded DNA viruses Edit

The first Baltimore group of DNA viruses are those that have a double-stranded DNA genome. All dsDNA viruses have their mRNA synthesized in a three-step process. First, a transcription preinitiation complex binds to the DNA upstream of the site where transcription begins, allowing for the recruitment of a host RNA polymerase. Second, once the RNA polymerase is recruited, it uses the negative strand as a template for synthesizing mRNA strands. Third, the RNA polymerase terminates transcription upon reaching a specific signal, such as a polyadenylation site. [2] [3] [4]

dsDNA viruses make use of several mechanisms to replicate their genome. Bidirectional replication, in which two replication forks are established at a replication origin site and move in opposite directions of each other, is widely used. [5] A rolling circle mechanism that produces linear strands while progressing in a loop around the circular genome is also common. [6] [7] Some dsDNA viruses use a strand displacement method whereby one strand is synthesized from a template strand, and a complementary strand is then synthesized from the prior synthesized strand, forming a dsDNA genome. [8] Lastly, some dsDNA viruses are replicated as part of a process called replicative transposition whereby a viral genome in a host cell's DNA is replicated to another part of a host genome. [9]

dsDNA viruses can be subdivided between those that replicate in the nucleus, and as such are relatively dependent on host cell machinery for transcription and replication, and those that replicate in the cytoplasm, in which case they have evolved or acquired their own means of executing transcription and replication. [10] dsDNA viruses are also commonly divided between tailed dsDNA viruses, referring to members of the realm Duplodnaviria, usually the tailed bacteriophages of the order Caudovirales, and tailless or non-tailed dsDNA viruses of the realm Varidnaviria. [11] [12]

Single-stranded DNA viruses Edit

The second Baltimore group of DNA viruses are those that have a single-stranded DNA genome. ssDNA viruses have the same manner of transcription as dsDNA viruses. However, because the genome is single-stranded, it is first made into a double-stranded form by a DNA polymerase upon entering a host cell. mRNA is then synthesized from the double-stranded form. The double-stranded form of ssDNA viruses may be produced either directly after entry into a cell or as a consequence of replication of the viral genome. [13] [14] Eukaryotic ssDNA viruses are replicated in the nucleus. [10] [15]

Most ssDNA viruses contain circular genomes that are replicated via rolling circle replication (RCR). ssDNA RCR is initiated by an endonuclease that bonds to and cleaves the positive strand, allowing a DNA polymerase to use the negative strand as a template for replication. Replication progresses in a loop around the genome by means of extending the 3'-end of the positive strand, displacing the prior positive strand, and the endonuclease cleaves the positive strand again to create a standalone genome that is ligated into a circular loop. The new ssDNA may be packaged into virions or replicated by a DNA polymerase to form a double-stranded form for transcription or continuation of the replication cycle. [13] [16]

Parvoviruses contain linear ssDNA genomes that are replicated via rolling hairpin replication (RHR), which is similar to RCR. Parvovirus genomes have hairpin loops at each end of the genome that repeatedly unfold and refold during replication to change the direction of DNA synthesis to move back and forth along the genome, producing numerous copies of the genome in a continuous process. Individual genomes are then excised from this molecule by the viral endonuclease. For parvoviruses, either the positive or negative sense strand may be packaged into capsids, varying from virus to virus. [16] [17]

Nearly all ssDNA viruses have positive sense genomes, but a few exceptions and peculiarities exist. The family Anelloviridae is the only ssDNA family whose members have negative sense genomes, which are circular. [15] Parvoviruses, as previously mentioned, may package either the positive or negative sense strand into virions. [14] Lastly, bidnaviruses package both the positive and negative linear strands. [15] [18]

The International Committee on Taxonomy of Viruses (ICTV) oversees virus taxonomy and organizes viruses at the basal level at the rank of realm. Virus realms correspond to the rank of domain used for cellular life but differ in that viruses within a realm do not necessarily share common ancestry, nor do the realms share common ancestry with each other. As such, each virus realm represents at least one instance of viruses coming into existence. Within each realm, viruses are grouped together based on shared characteristics that are highly conserved over time. [19] Three DNA virus realms are recognized: Duplodnaviria, Monodnaviria, and Varidnaviria.

Duplodnaviria Edit

Duplodnaviria contains dsDNA viruses that encode a major capsid protein (MCP) that has the HK97 fold. Viruses in the realm also share a number of other characteristics involving the capsid and capsid assembly, including an icosahedral capsid shape and a terminase enzyme that packages viral DNA into the capsid during assembly. Two groups of viruses are included in the realm: tailed bacteriophages, which infect prokaryotes and are assigned to the order Caudovirales, and herpesviruses, which infect animals and are assigned to the order Herpesvirales. [11]

Duplodnaviria is either monophyletic or polyphyletic and may predate the last universal common ancestor (LUCA) of cellular life. The exact origin of the realm is not known, but the HK97-fold found in the MCP of all members is, outside the realm, only found in encapsulins, a type of nanocompartment found in bacteria, although the relation between Duplodnaviria and encapsulins is not fully understood. [11] [20] [21]

The relation between caudoviruses and herpesviruses is not certain, as they may either share a common ancestor or herpesviruses may be a divergent clade from within Caudovirales. A common trait among duplodnaviruses is that they cause latent infections without replication while still being able to replicate in the future. [22] [23] Tailed bacteriophages are ubiquitous worldwide, [24] important in marine ecology, [25] and the subject of much research. [26] Herpesviruses are known to cause a variety of epithelial diseases, including herpes simplex, chickenpox and shingles, and Kaposi's sarcoma. [27] [28] [29]

Monodnaviria Edit

Monodnaviria contains ssDNA viruses that encode an endonuclease of the HUH superfamily that initiates rolling circle replication and all other viruses descended from such viruses. The prototypical members of the realm are called CRESS-DNA viruses and have circular ssDNA genomes. ssDNA viruses with linear genomes are descended from them, and in turn some dsDNA viruses with circular genomes are descended from linear ssDNA viruses. [30]

Viruses in Monodnaviria appear to have emerged on multiple occasions from archaeal and bacterial plasmids, a type of extra-chromosomal DNA molecule that self-replicates inside its host. The kingdom Shotokuvirae in the realm likely emerged from recombination events that merged the DNA of these plasmids and complementary DNA encoding the capsid proteins of RNA viruses. [30] [31]

CRESS-DNA viruses include three kingdoms that infect prokaryotes: Loebvirae, Sangervirae, and Trapavirae. The kingdom Shotokuvirae contains eukaryotic CRESS-DNA viruses and the atypical members of Monodnaviria. [30] Eukaryotic monodnaviruses are associated with many diseases, and they include papillomaviruses and polyomaviruses, which cause many cancers, [32] [33] and geminiviruses, which infect many economically important crops. [34]

Varidnaviria Edit

Varidnaviria contains DNA viruses that encode MCPs that have a jelly roll fold folded structure in which the jelly roll (JR) fold is perpendicular to the surface of the viral capsid. Many members also share a variety of other characteristics, including a minor capsid protein that has a single JR fold, an ATPase that packages the genome during capsid assembly, and a common DNA polymerase. Two kingdoms are recognized: Helvetiavirae, whose members have MCPs with a single vertical JR fold, and Bamfordvirae, whose members have MCPs with two vertical JR folds. [12]

Varidnaviria is either monophyletic or polyphyletic and may predate the LUCA. The kingdom Bamfordvirae is likely derived from the other kingdom Helvetiavirae via fusion of two MCPs to have an MCP with two jelly roll folds instead of one. The single jelly roll (SJR) fold MCPs of Helvetiavirae show a relation to a group of proteins that contain SJR folds, including the Cupin superfamily and nucleoplasmins. [12] [20] [21]

Marine viruses in Varidnaviria are ubiquitous worldwide and, like tailed bacteriophages, play an important role in marine ecology. [35] Most identified eukaryotic DNA viruses belong to the realm. [36] Notable disease-causing viruses in Varidnaviria include adenoviruses, poxviruses, and the African swine fever virus. [37] Poxviruses have been highly prominent in the history of modern medicine, especially Variola virus, which caused smallpox. [38] Many varidnaviruses are able to become endogenized in their host's genome, and a peculiar example of this are virophages, which confer protection for their hosts against giant viruses during infection. [36]

By Baltimore group Edit

dsDNA viruses are classified into three realms and include many taxa that are unassigned to a realm:

  • All viruses in Duplodnaviria are dsDNA viruses. [11]
  • In Monodnaviria, members of the class Papovaviricetes are dsDNA viruses. [30]
  • All viruses in Varidnaviria are dsDNA viruses. [12]
  • The following taxa that are unassigned to a realm exclusively contain dsDNA viruses: [12]
    • Orders: Ligamenvirales
    • Families: Ampullaviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Halspiviridae, Hytrosaviridae, Nimaviridae, Nudiviridae, Ovaliviridae, Plasmaviridae, Polydnaviridae, Portogloboviridae, Thaspiviridae, Tristromaviridae
    • Genera: Dinodnavirus, Rhizidiovirus

    ssDNA viruses are classified into one realm and include several families that are unassigned to a realm:


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