A New Rodent Model to Understand Host-Virus Interactions at the Early Stages of Retroviral Endogenization

Unlike other viruses, retroviruses replicate by copying their RNA genomes to DNA. They therefore become a largely inseparable part of the cell's genome upon infection. Retroviruses can be transmitted horizontally and vertically by infection and often have wide cellular tropism. Exogenous retroviral infection (XRV) occurs in somatic cells, but when infection is in the germline, the resulting provirus is known as an endogenous retrovirus (ERVs). Accumulation of these retroviral sequences over evolutionary time has granted them ~ 8% occupancy of the human genome and, along with other transposable elements (TEs), makes them a major determinant of DNA sequence diversity and driver of species evolution. Millions of years of evolution have obscured the history of mutation, indel, rearrangement and distribution events that ERVs have experienced since they integrated. Understanding how ERVs establish themselves in a host genome is crucial to infer vertebrate adaptive immunity and the generated memory of these genome invaders. Koala retrovirus (KoRV), as the only known mammalian retrovirus currently undergoing genome colonization, is generally used as a model system for mechanism of endogenization. However the precursor vector species that gave rise to KoRV and the closely related pathogenic Gibbon Ape Leukemia Virus (GALV) remains obscure. In an attempt to identify the reservoir of GALV-KoRV, we have identified a novel infectious GALV virus in a specific population of a native rodent of Papua New Guinea, Melomys leucogaster. We named this virus, complete melomys woolly monkey virus (cMWMV). Using cell culture methods, fluorescence, and electron microscopy, we have characterized this gammaretrovirus. The significance of cMWMV is that like KoRV, it is currently invading the genome of a new host species. As KoRV is restricted to koalas, cMWMV could provide an additional rodent model to further study the evolutionary processes that contribute to the germline invasion and adaptation to a new host. This recent retroviral invasion can help us elucidate the general principles of antiretroviral gene evolution within Melomys and between rodent species that are known to be under diversifying selection in the primate orthologs. PacBio sequencing was used to sequence the whole genome of Melomys. Guided sequence alignment was performed and exons corresponding to genes of interest were extracted. Coding sequences (CDS) were de novo assembled and manually curated. We then used various substitution models to quantify the selection pressure in these immune genes. Our data suggest that, similar to primates, these genes may have experienced positive selection at some sites (codons) in Mus musculus and Rattus norvegicus lineages. However the excess of synonymous sites asserts a long-term trend of purifying selection. A weak intensified diversifying selection pattern in Melomys lineage of ZAP (zinc-finger CCCH-type antiviral protein 1) gene could indicate an effort to inhibit viral mRNA translation of the endogenizing cMWMV.Im Gegensatz zu anderen Viren replizieren sich Retroviren, indem sie ihr RNS-Genom in DNS kopieren. Daher werden sie nach der Infektion zu einem weitgehend untrennbaren Teil des Zellgenoms. Retroviren können durch Infektion horizontal und vertikal übertragen werden und haben oft einen breiten Zelltropismus. Eine exogene retrovirale Infektion (XRV) findet in somatischen Zellen statt. Erfolgt jedoch die Infektion in der Keimbahn, wird das resultierende Provirus als endogenes Retrovirus (ERV) bezeichnet. Die Anhäufung dieser retroviralen Sequenzen im Laufe der Evolution hat dazu geführt, dass sie ca. 8 % des menschlichen Genoms einnehmen und zusammen mit anderen transponierbaren Elementen (TEs) eine wichtige Determinante der DNS-Sequenzvielfalt sowie eine treibende Kraft für die Evolution der Arten darstellen. Jahrmillionen der Evolution haben den Verlauf von Mutation, Indel, Umlagerung und Verbreitung, die ERVs seit ihrer Integration erfahren haben, verschleiert. Die Art und Weise, wie sich ERVs in einem Wirtsgenom etablieren, ist entscheidend, um Rückschlüsse auf die adaptive Immunität von Wirbeltieren und das erzeugte Gedächtnis dieser Genom-Invasoren zu ziehen. Das Koala-Retrovirus (KoRV), das einzige bekannte Säugetier-Retrovirus, das derzeit eine Genomkolonisierung durchläuft, wird im Allgemeinen als Modellsystem für den Mechanismus der Endogenisierung verwendet. Die Vorläufer-Vektorspezies, die KoRV und das eng verwandte pathogene Gibbon-Affen-Leukämievirus (GALV) hervorgebracht hat, ist jedoch nach wie vor unbekannt. In einem Versuch, das Reservoir von GALV-KoRV zu identifizieren, haben wir ein neuartiges infektiöses GALV-Virus in einer bestimmten Population eines in Papua-Neuguinea heimischen Nagetiers, Melomys leucogaster, nachgewiesen. Das Virus wurde complete Melomys Woolly Monkey Virus (cMWMV) genannt. Mit Hilfe von Zellkulturmethoden, Fluoreszenz- und Elektronenmikroskopie haben wir dieses Gammaretrovirus charakterisiert. Die Besonderheit von cMWMV besteht darin, dass es, wie KoRV, derzeit in das Genom einer neuen Wirtsart eindringt. Da KoRV nur bei Koalas vorkommt, könnte cMWMV ein zusätzliches Nagetiermodell sein, um die evolutionären Prozesse zu untersuchen, die zur Keimbahninvasion und Anpassung an einen neuen Wirt beitragen. Diese jüngste retrovirale Invasion kann uns helfen, die allgemeinen Prinzipien der antiretroviralen Genevolution innerhalb von Melomys und zwischen Nagetierarten zu verdeutlichen, die bekanntermaßen der diversifizierenden Selektion der Primatenorthologen unterliegen. Mittels PacBio-Sequenzierung wurde das gesamte Genom von Melomys sequenziert. Ein geführter Sequenzabgleich wurde vorgenommen und die den relevanten Genen entsprechenden Exone extrahiert. Die kodierenden Sequenzen (CDS) wurden de novo assembliert und manuell kuratiert. Anschließend haben wir verschiedene Substitutionsmodelle angewandt, um den Selektionsdruck in diesen Immungenen zu quantifizieren. Unsere Daten deuten darauf hin, dass diese Gene, ähnlich wie bei den Primaten, in den Abstammungslinien von Mus musculus und Rattus norvegicus an einigen Stellen (Codons) einer positiven Selektion unterlegen haben könnten. Der Überschuss an synonymen Stellen deutet jedoch auf einen langfristigen Trend der reinigenden Selektion hin. Ein schwaches, verstärkt diversifizierendes Selektionsmuster in der Melomys-Abstammungslinie des ZAP-Gens (Zink-Finger-CCCH-Typ antivirales Protein 1) könnte auf einen Versuch hindeuten, die virale mRNA-Translation des endogenisierenden cMWMV zu inhibieren

A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization’s occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered . The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace’s Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents ( Melomys ) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease

The SARS-CoV-2 main protease (Mpro) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain required for tRNA modification activity in cells. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 may be resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. We determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage showed that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage that could help inform future therapeutic design and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis. Viral proteases can strategically target human proteins to manipulate host biochemistry during infection. Here, we show that the SARS-CoV-2 main protease (Mpro) can specifically recognize and cleave the human tRNA methyltransferase enzyme TRMT1, which installs a modification on human tRNAs that is critical for protein translation. Our structural and functional analysis of the Mpro-TRMT1 interaction shows how the flexible Mpro active site engages a conserved sequence in TRMT1 in an uncommon binding mode to catalyze its cleavage and inactivation. These studies provide new insights into substrate recognition by SARS-CoV-2 Mpro that could inform future antiviral therapeutic design and suggest that proteolysis of TRMT1 during SARS-CoV-2 infection may disrupt tRNA modification and host translation to impact COVID-19 pathogenesis or phenotypes