Rapid Accumulation Of Mutations Research Articles

Suicidal Phenotype of Proofreading-Deficient Herpes Simplex Virus 1 Polymerase Mutants.

Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.

Impact of SARS-CoV-2 Mutations on PCR Assay Sequence Alignment.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.

Evidence for a mouse origin of the SARS-CoV-2 Omicron variant

The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting a possibility of host-jumping. The molecular spectrum of mutations (i.e., the relative frequency of the 12 types of base substitutions) acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients but resembled the spectra associated with virus evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.

Sequential Immunizations with heterosubtypic virus-like particles elicit cross protection against divergent influenza A viruses in mice

Seasonal influenza vaccines have proven to be effective against well-matched viruses in healthy adults. However, rapid accumulation of mutations in the main antigenic surface proteins of influenza can compromise the efficiency of flu vaccines. Occasionally, influenza pandemics arise and present a different type of challenge to current seasonal vaccines. Novel vaccination strategies that can educate the host immune system to generate immune responses focusing on conserved epitopes on theses antigenic surface proteins are crucial for controlling and limiting influenza epidemics and pandemics. In this study, we have sequentially vaccinated mice with heterosubtypic influenza HA virus-like particles (VLPs) harboring H1, H8, and H13 from the HA phylogenetic group 1, or H3, H4, and H10 from the HA phylogenetic group 2, or in various combinations. The immunized animals were fully protected when challenged with lethal doses of heterosubtypic viruses from either phylogenetic group. Our vaccination approach demonstrates a promising strategy for the development of a ‘universal influenza vaccine’.

New insights into the evolutionary rate of hepatitis B virus at different biological scales.

The evolutionary rates of hepatitis B virus (HBV) estimated using contemporary sequences are 10(2) to 10(4) times higher than those derived from archaeological and genetic evidence. This discrepancy makes the origin of HBV and the time scale of its spread, both of which are critical for studying the burden of HBV pathogenicity, largely unresolved. To evaluate whether the dual demands (i.e., adaptation within hosts and colonization between hosts) of the viral life cycle affect this conundrum, the HBV quasispecies dynamics within and among hosts from a family consisting of a grandmother, her 5 children, and her 2 granddaughters, all of whom presumably acquired chronic HBV through mother-to-infant transmission, were examined by PCR cloning and next-generation sequencing methods. We found that the evolutionary rate of HBV between hosts was considerably lower than that within hosts. Moreover, the between-host substitution rates of HBV decreased as transmission numbers between individuals increased. Both observations were due primarily to changes at nonsynonymous rather than synonymous sites. There were significantly more multiple substitutions than expected for random mutation processes, and 97% of substitutions were changed from common to rare amino acid residues in the database. Continual switching between colonization and adaptation resulted in a rapid accumulation of mutations at a limited number of positions, which quickly became saturated, whereas substitutions at the remaining regions occurred at a much lower rate. Our study may help to explain the time-dependent HBV substitution rates reported in the literature and provide new insights into the origin of the virus. It is known that the estimated hepatitis B virus (HBV) substitution rate is time dependent, but the reason behind this observation is still elusive. We hypothesize that owing to the small genome size of HBV, transmission between hosts and adaptation within hosts must exhibit high levels of fitness trade-offs for the virus. By studying the HBV quasispecies dynamics for a chain of sequentially infected transmissions within a family, we found the HBV substitution rate between patients to be negatively correlated with the number of transmissions. Continual switching between hosts resulted in a rapid accumulation of mutations at a limited number of genomic sites, which quickly became saturated in the short term. Nevertheless, substitutions at the remaining regions occurred at a much lower rate. Therefore, the HBV substitution rate decreased as the divergence time increased.

Tick-borne encephalitis virus subtypes emerged through rapid vector switches rather than gradual evolution.

Tick-borne encephalitis is the most important human arthropod-borne virus disease in Europe and Russia, with an annual incidence of about 13 thousand people. Tick-borne encephalitis virus (TBEV) is distributed in the natural foci of forest and taiga zones of Eurasia, from the Pacific to the Atlantic coast. Currently, there are three mutually exclusive hypotheses about the origin and distribution of TBEV subtypes, although they are based on the same assumption of gradual evolution. Recently, we have described the structure of TBEV populations in terms of a clusteron approach, a clusteron being a structural unit of viral population [Kovalev and Mukhacheva (2013) Infect. Genet. Evol., 14, 22–28]. This approach allowed us to investigate questions of TBEV evolution in a new way and to propose a hypothesis of quantum evolution due to a vector switch. We also consider a possible mechanism for this switch occurring in interspecific hybrids of ticks. It is necessarily accompanied by a rapid accumulation of mutations in the virus genome, which is contrary to the generally accepted view of gradual evolution in assessing the ages of TBEV populations. The proposed hypothesis could explain and predict not only the formation of new subtypes, but also the emergence of new vector-borne viruses.

Obligate Insect Endosymbionts Exhibit Increased Ortholog Length Variation and Loss of Large Accessory Proteins Concurrent with Genome Shrinkage

Extreme genome reduction has been observed in obligate intracellular insect mutualists and is an assumed consequence of fixed, long-term host isolation. Rapid accumulation of mutations and pseudogenization of genes no longer vital for an intracellular lifestyle, followed by deletion of many genes, are factors that lead to genome reduction. Size reductions in individual genes due to small-scale deletions have also been implicated in contributing to overall genome shrinkage. Conserved protein functional domains are expected to exhibit low tolerance for mutations and therefore remain relatively unchanged throughout protein length reduction while nondomain regions, presumably under less selective pressures, would shorten. This hypothesis was tested using orthologous protein sets from the Flavobacteriaceae (phylum: Bacteroidetes) and Enterobacteriaceae (subphylum: Gammaproteobacteria) families, each of which includes some of the smallest known genomes. Upon examination of protein, functional domain, and nondomain region lengths, we found that proteins were not uniformly shrinking with genome reduction, but instead increased length variability and variability was observed in both the functional domain and nondomain regions. Additionally, as complete gene loss also contributes to overall genome shrinkage, we found that the largest proteins in the proteomes of nonhost-restricted bacteroidetial and gammaproteobacterial species often were inferred to be involved in secondary metabolic processes, extracellular sensing, or of unknown function. These proteins were absent in the proteomes of obligate insect endosymbionts. Therefore, loss of genes encoding large proteins not required for host-restricted lifestyles in obligate endosymbiont proteomes likely contributes to extreme genome reduction to a greater degree than gene shrinkage.

Abstract 1752: Consequences of high Chromosome Instability (CIN) in SGO1(Shugoshin1) CIN model mice.

Abstract Abstract Mitotic errors have been suspected to be a cause of aneuploidy, rapid accumulation of mutations, and tumorigenesis. In addition, recent cell biological studies demonstrated that mitotic errors can result in DNA damage, which would also aid accumulation of mutations then carcinogenesis. However, no observations in vivo have been provided to support the cell biological observations to date. Previously we developed two strains of high Chromosome Instability (CIN) model mice, which show an increase in mitotic errors due to haploinsufficiency in chromosome cohesion system component Sgo1 or mitotic spindle checkpoint component BubR1. The pathways affected by these mutations are suggested to play a role in aging and carcinogenesis in human. Consistently, compared with wild type, both mice strains showed a significant risk for tumor development in colon with chemical carcinogenesis assays. In this study, we used these CIN mice to test whether (i) spontaneous carcinogenesis occurs in the mice, (ii) spontaneous DNA damage occurs in the mice, (iii) whether associated marker expressions are altered, and (iv) if so, whether it is affected by the age. In 12 month old (middle-age) SGO1 and BUBR1 mice, we observed an increase in spontaneous epithelial cancers, although the carcinogenic effect is not aggressive (p<0.05 in SGO1). In histologically normal-looking liver tissue in the mice, increased signals of DNA damage marker pohspho-gamma-H2AX and p53 were observed (4∼5-fold higher in CIN mice), along with altered expressions of p16INK4A and Bcl2 (2∼6-fold higher in CIN mice), providing the first evidence for DNA damage in vivo with mutations that increase mitotic errors. In 4 month old (younger adult) SGO1 mice, significantly (p<0.05) higher percentage of p-H2AX signal in comparison with wild type was also observed. The results demonstrate that mitotic error and CIN-causing mutation can cause higher DNA damage in the tissue-level, indicating that an additional path to carcinogenesis is at work in vivo. Marker analysis in the CIN model mice would indicate molecular consequences of this type of mutation and would lead to the discovery of novel cancer prevention drug targets. Citation Format: Hiroshi Y. Yamada, Yuting Zhang, Wei Dai, Chinthalapally V. Rao. Consequences of high Chromosome Instability (CIN) in SGO1(Shugoshin1) CIN model mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1752. doi:10.1158/1538-7445.AM2013-1752

Dynamic transposable element accumulation in the nascent sex chromosomes of papaya

From their inception, Y chromosomes in plants and animals are subjected to the powerful effects of Müller’s ratchet, a process spurred by suppression of recombination that results in a rapid accumulation of mutations and repetitive elements. These mutations eventually lead to gene loss and degeneration of the Y chromosome. Y chromosomes in mammals are ancient, whereas most sex chromosomes in plants and many in insects and fish evolved recently. Sex type in papaya is controlled by a pair of nascent sex chromosomes that evolved around 7 million years ago. The papaya X and Yh were recently sequenced, providing valuable insight into the early stages of sex chromosome evolution. Here we discuss the fruits of this work with a focus on the repeat accumulation, gene trafficking and promiscuous DNA sequences found in the slowly degenerating Yh chromosome of papaya.

Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

Sequence of PRAT satellite DNA "frozen" in some Coleopteran species.

The intriguing diversity of highly abundant satellite repeats found even among closely related species can result from processes leading to dramatic changes in copy number of a particular sequence in the genome and not from rapid accumulation of mutations. To test this hypothesis, we investigated the distribution of the PRAT satellite DNA family, a highly abundant major satellite in the coleopteran species Palorus ratzeburgii, in eight species belonging to the related genera ( Tribolium, Tenebrio, Latheticus), the subfamily (Pimeliinae), and the family (Chrysomelidae). Dot blot analysis and PCR assay followed by Southern hybridization revealed that the PRAT satellite, in the form of low-copy number repeats, was present in all tested species. The PRAT satellite detected in the species Pimelia elevata has been sequenced, and compared with previously cloned PRAT monomers from Palorus ratzeburgii and Palorus subdepressus. Although the two Palorus species diverged at least 7 Myr ago, and the subfamily Pimeliinae separated from the genus Palorus 50-60 Myr ago, all PRAT clones exhibit high mutual homology, with average variability relative to the common consensus sequence of 1.3%. The presence of ancestral mutations found in PRAT clones from all three species as well as the absence of species diagnostic mutations illustrate extremely slow sequence evolution. This unexpectedly high conservation of PRAT satellite DNA sequence might be induced by a small bias of turnover mechanisms favoring the ancestral sequence in the process of molecular drive.