Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

ABSTRACT The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication: initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function. IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

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Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

Frontiers in Microbiology ◽

10.3389/fmicb.2021.738125 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lisett Liblekas ◽

Alla Piirsoo ◽

Annika Laanemets ◽

Eva-Maria Tombak ◽

Airiin Laaneväli ◽

...

Keyword(s):

Life Cycle ◽

Viral Genome ◽

Maintenance Phase ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Genome Replication ◽

Viral Genomes ◽

Viral Genome Replication ◽

Infected Cells ◽

Cell Genome

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

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Dual Role of a Viral Polymerase in Viral Genome Replication and Particle Self-Assembly

mBio ◽

10.1128/mbio.01242-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 2

Author(s):

Xiaoyu Sun ◽

Serban L. Ilca ◽

Juha T. Huiskonen ◽

Minna M. Poranen

Keyword(s):

Self Assembly ◽

Viral Genome ◽

Rna Viruses ◽

The Self ◽

Genome Replication ◽

Double Stranded Rna ◽

Polymerase Complex ◽

Viral Genome Replication ◽

Dsrna Viruses

ABSTRACTDouble-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilizedPseudomonasphage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions.IMPORTANCEDouble-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly.

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Hitchhiking of Viral Genomes on Cellular Chromosomes

Annual Review of Virology ◽

10.1146/annurev-virology-092818-015716 ◽

2019 ◽

Vol 6 (1) ◽

pp. 275-296 ◽

Cited By ~ 3

Author(s):

Tami L. Coursey ◽

Alison A. McBride

Keyword(s):

Viral Genome ◽

Viral Infections ◽

Genome Replication ◽

Mitotic Chromosomes ◽

Dna Viruses ◽

Viral Genomes ◽

Barr Virus ◽

Dividing Cells ◽

Viral Genome Replication ◽

Epstein Barr

Persistent viral infections require a host cell reservoir that maintains functional copies of the viral genome. To this end, several DNA viruses maintain their genomes as extrachromosomal DNA minichromosomes in actively dividing cells. These viruses typically encode a viral protein that binds specifically to viral DNA genomes and tethers them to host mitotic chromosomes, thus enabling the viral genomes to hitchhike or piggyback into daughter cells. Viruses that use this tethering mechanism include papillomaviruses and the gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This review describes the advantages and consequences of persistent extrachromosomal viral genome replication.

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In VivoLabelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication

Journal of Virology ◽

10.1128/jvi.00795-18 ◽

2018 ◽

Vol 92 (18) ◽

Cited By ~ 19

Author(s):

Tetsuro Komatsu ◽

Charlotte Quentin-Froignant ◽

Irene Carlon-Andres ◽

Floriane Lagadec ◽

Fabienne Rayne ◽

...

Keyword(s):

Life Cycle ◽

Real Time ◽

Viral Genome ◽

Imaging System ◽

Morphological Changes ◽

Host Cells ◽

Genome Replication ◽

Equal Distribution ◽

Viral Genomes

ABSTRACTAdenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatilein vivoimaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction within vivogenome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomesin vivo. The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase.IMPORTANCEViruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, anin vivoDNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted thein vivovisualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.

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The Role of DNA Repair Pathways in Adeno-Associated Virus Infection and Viral Genome Replication / Recombination / Integration

DNA Repair and Human Health ◽

10.5772/24265 ◽

2011 ◽

Cited By ~ 1

Author(s):

Kei Adachi ◽

Hiroyuki Nakai

Keyword(s):

Dna Repair ◽

Virus Infection ◽

Viral Genome ◽

Genome Replication ◽

Adeno Associated Virus ◽

Viral Genome Replication ◽

Repair Pathways

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Inducible heat shock protein 70 enhances HPV31 viral genome replication and virion production during the differentiation-dependent life cycle in human keratinocytes

Virus Research ◽

10.1016/j.virusres.2009.10.019 ◽

2010 ◽

Vol 147 (1) ◽

pp. 113-122 ◽

Cited By ~ 16

Author(s):

Hebin Song ◽

Pope L. Moseley ◽

Stephanie L. Lowe ◽

Michelle A. Ozbun

Keyword(s):

Life Cycle ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Viral Genome ◽

Human Keratinocytes ◽

Genome Replication ◽

Virion Production ◽

Viral Genome Replication

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Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein

Journal of Virology ◽

10.1128/jvi.02642-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4153-4162 ◽

Cited By ~ 68

Author(s):

Zejun Li ◽

Tokiko Watanabe ◽

Masato Hatta ◽

Shinji Watanabe ◽

Asuka Nanbo ◽

...

Keyword(s):

Amino Acids ◽

Life Cycle ◽

Viral Replication ◽

Viral Genome ◽

Influenza A ◽

Mutational Analysis ◽

Viral Rna ◽

Genome Replication ◽

Viral Genome Replication ◽

Conserved Amino Acids

ABSTRACT The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

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Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

mBio ◽

10.1128/mbio.03315-21 ◽

2022 ◽

Author(s):

Sho Miyamoto ◽

Masahiro Nakano ◽

Takeshi Morikawa ◽

Ai Hirabayashi ◽

Ryoma Tamura ◽

...

Keyword(s):

Influenza Virus ◽

Life Cycle ◽

Viral Genome ◽

Influenza A ◽

Genome Replication ◽

Ribonucleoprotein Complex ◽

Virus Life Cycle ◽

Viral Genome Replication ◽

Infected Cells ◽

Nuclear Domains

Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process.

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Alternative translation and alternative RNA processing mechanism in parvovirus RNA processing

10.32469/10355/45905 ◽

2014 ◽

Author(s):

◽

Olufemi Fasina

Keyword(s):

Gene Expression ◽

Rna Processing ◽

Viral Genome ◽

Transcription Initiation ◽

Alternative Polyadenylation ◽

Open Reading Frames ◽

Genome Replication ◽

University Of Missouri ◽

Diverse Range ◽

Viral Genome Replication

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Viruses as obligate intracellular metabolic parasite require the capacity to orchestrate and modulate the host environment either in the nucleus or cytoplasm for their efficient reproductive life cycle. This warrants the use of diverse range of proteins expressed from the viral genome with the ability of regulating viral genome replication, transcription and translation, in addition antagonizing host factors inhibitory to the virus. Therefore, in order to achieve these goals, viruses utilizes gene expression strategies to expand their coding capacity. Gene expression mechanism such as transcription initiation, capping, splicing and 3ï¿½-end processing afford viruses the opportunities to utilize the eukaryotic metabolic machineries for generating proteome diversity. Parvoviruses and other DNA viruses effectively capitalize on their use of nuclear eukaryotic metabolic machineries to co-opt host cell factors for optimal replication and gene expression. Parvoviruses with small genome size and overlapping open reading frames utilize alternative transcription initiation, alternative splicing and alternative polyadenylation to co-ordinate the expression of its non-structural and structural proteins. In this work, we have characterized how two parvoviruses; Dependovirus AAV5 and Bocavirus Minute virus of canine (MVC) utilize alternative gene expression mechanisms and strategies to optimize expression of viral proteins from their genome.

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