scholarly journals Persistence of an Oncogenic Papillomavirus Genome RequirescisElements from the Viral Transcriptional Enhancer

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Koenraad Van Doorslaer ◽  
Dan Chen ◽  
Sandra Chapman ◽  
Jameela Khan ◽  
Alison A. McBride
Keyword(s):  
Persistent Infection ◽  
Viral Genome ◽  
Replication Origin ◽  
Human Papillomaviruses ◽  
Wild Type ◽  
Transcriptional Enhancer ◽  
Complementation Assay ◽  
Viral Genomes ◽  
Stable Maintenance

ABSTRACTHuman papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viraltransfactors andciselements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins intrans, allowing us to determine additionalciselements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required incisfor long-term genome replication.IMPORTANCEHuman papillomaviruses infect cutaneous and mucosal epithelial cells of the host, and this results in very-long-lived, persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA. This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. This has been difficult to study, because viral transcription, replication, and partitioning are regulated by the same viral proteins and involve overlapping elements in the viral genome. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome. This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, thus curing persistent HPV infection.

Regulation of HPV18 Genome Replication, Establishment and Persistence by Sequences in the Viral Upstream Regulatory Region

2021 ◽  
Author(s):  
Tami L. Coursey ◽  
Koenraad Van Doorslaer ◽  
Alison A. McBride
Keyword(s):  
Replication Origin ◽  
Regulatory Region ◽  
Unique Element ◽  
Genome Replication ◽  
Transcriptional Enhancer ◽  
Viral Genomes ◽  
Daughter Cells ◽  
Papillomavirus Infection ◽  
Stable Dna Replication

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses. Importance Persistent infection with oncogenic HPVs is responsible for ∼5% human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of anti-viral therapies.

scholarly journals A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

2008 ◽  
Vol 82 (17) ◽  
pp. 8500-8508 ◽  
Author(s):  
Haiyan Li ◽  
Kazufumi Ikuta ◽  
John W. Sixbey ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Viral Genome ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

scholarly journals Systemic Polyomavirus Genome Increase and Dissemination of Capsid-Defective Genomes in Mammary Gland Tumor-Bearing Mice

2000 ◽  
Vol 74 (15) ◽  
pp. 6975-6983 ◽  
Author(s):  
Julie J. Wirth ◽  
Li Chen ◽  
Michele M. Fluck
Keyword(s):  
Mammary Gland ◽  
Viral Genome ◽  
Wild Type Strain ◽  
Neonatal Infection ◽  
Wild Type ◽  
Coding Region ◽  
Live Virus ◽  
Viral Genomes ◽  
Mammary Gland Tumors ◽  
Low Levels

ABSTRACT BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.

scholarly journals Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Bora Nam ◽  
Zelalem Mekuria ◽  
Mariano Carossino ◽  
Ganwu Li ◽  
Ying Zheng ◽  
...  
Keyword(s):  
Persistent Infection ◽  
Viral Genome ◽  
Reproductive Tract ◽  
Purifying Selection ◽  
Buffy Coat ◽  
Equine Arteritis Virus ◽  
Nucleotide Substitutions ◽  
Genome Wide ◽  
Nasal Secretions

ABSTRACTEquine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCEEAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

scholarly journals The evolution of collective infectious units in viruses

2019 ◽  
Author(s):  
Asher Leeks ◽  
Rafael Sanjuán ◽  
Stuart A. West
Keyword(s):  
Viral Genome ◽  
Social Evolution ◽  
Viral Pathogenesis ◽  
Wild Type Virus ◽  
Antiviral Resistance ◽  
Costs And Benefits ◽  
Wild Type ◽  
Viral Genomes ◽  
Viral Sequences ◽  
Infectious Unit

Viruses frequently spread among cells or hosts in groups, with multiple viral genomes inside the same infectious unit. These collective infectious units can consist of multiple viral genomes inside the same virion, or multiple virions inside a larger structure such as a vesicle. Collective infectious units deliver multiple viral genomes to the same cell simultaneously, which can have important implications for viral pathogenesis, antiviral resistance, and social evolution. However, little is known about why some viruses transmit in collective infectious units, whereas others do not. We used a simple evolutionary approach to model the potential costs and benefits of transmitting in a collective infectious unit. We found that collective infectious units could be favoured if cells infected by multiple viral genomes were significantly more productive than cells infected by just one viral genome, and especially if there were also efficiency benefits to packaging multiple viral genomes inside the same infectious unit. We also found that if some viral sequences are defective, then collective infectious units could evolve to become very large, but that if these defective sequences interfered with wild-type virus replication, then collective infectious units were disfavoured.

scholarly journals Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

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.

scholarly journals Sequences in the Human papillomavirus Type 18 (HPV18) Upstream Regulatory Region regulate Viral Genome Replication, Establishment and Persistence

2021 ◽  
Author(s):  
Tami L. Coursey ◽  
Koenraad Van Doorslaer ◽  
Alison A. McBride
Keyword(s):  
Human Papillomavirus ◽  
Viral Genome ◽  
Replication Origin ◽  
Regulatory Region ◽  
Unique Element ◽  
Genome Replication ◽  
Transcriptional Enhancer ◽  
Papillomavirus Infection ◽  
Viral Genome Replication ◽  
Stable Dna Replication

AbstractDuring persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

scholarly journals SARS-CoV-2 Sequence Characteristics of COVID-19 Persistence and Reinfection

2021 ◽  
Author(s):  
Manish C Choudhary ◽  
Charles R Crain ◽  
Xueting Qiu ◽  
William Hanage ◽  
Jonathan Z Li
Keyword(s):  
Persistent Infection ◽  
Viral Evolution ◽  
Phylogenetic Reconstruction ◽  
Geographic Region ◽  
Antibody Treatment ◽  
Viral Genomes ◽  
Monoclonal Antibody Treatment ◽  
Viral Sequences ◽  
Sequence Characteristics ◽  
Baseline Health

Abstract Background Both SARS-CoV-2 reinfection and persistent infection have been reported, but sequence characteristics in these scenarios have not been described. We assessed published cases of SARS-CoV-2 reinfection and persistence, characterizing the hallmarks of reinfecting sequences and the rate of viral evolution in persistent infection. Methods A systematic review of PubMed was conducted to identify cases of SARS-CoV-2 reinfection and persistence with available sequences. Nucleotide and amino acid changes in the reinfecting sequence were compared to both the initial and contemporaneous community variants. Time-measured phylogenetic reconstruction was performed to compare intra-host viral evolution in persistent SARS-CoV-2 to community-driven evolution. Results Twenty reinfection and nine persistent infection cases were identified. Reports of reinfection cases spanned a broad distribution of ages, baseline health status, reinfection severity, and occurred as early as 1.5 months or >8 months after the initial infection. The reinfecting viral sequences had a median of 17.5 nucleotide changes with enrichment in the ORF8 and N genes. The number of changes did not differ by the severity of reinfection and reinfecting variants were similar to the contemporaneous sequences circulating in the community. Patients with persistent COVID-19 demonstrated more rapid accumulation of sequence changes than seen with community-driven evolution with continued evolution during convalescent plasma or monoclonal antibody treatment. Conclusions Reinfecting SARS-CoV-2 viral genomes largely mirror contemporaneous circulating sequences in that geographic region, while persistent COVID-19 has been largely described in immunosuppressed individuals and is associated with accelerated viral evolution.

An ER–Golgi Tethering Factor SLOH4/MIP3 Is Involved in Long-term Heat Tolerance of Arabidopsis

2020 ◽  
Author(s):  
Kazuho Isono ◽  
Ryo Tsukimoto ◽  
Satoshi Iuchi ◽  
Akihisa Shinozawa ◽  
Izumi Yotsui ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Tolerance ◽  
Secretory Proteins ◽  
Wild Type ◽  
Additional Function ◽  
Transcript Levels ◽  
Unfolded Protein ◽  
In The Wild ◽  
Protein Response

Abstract Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screening for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L- but not S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2, and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1–1 mutants was similar to that of the wild type. Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the wild type. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1)—accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins—were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not bZIP28, resulting in initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER–Golgi vesicle tethering.

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