Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Species-specific KRAB-ZFPs function as repressors of retroviruses by targeting PBS regions.

SignificanceHosts often target the relatively conserved regions in rapidly mutating retroviruses to inhibit their replication. One of these regions is called a primer binding site (PBS), which has to be complementary to the host tRNA to initiate reverse transcription. By analyzing endogenous retroviral elements, we found that host cells use this sequence as a target in efforts to block the expression of viral elements. A specific type of zinc finger protein targets the PBS in a host genome, which not only inhibits the transcription of endogenous viruses but also inhibits the replication of exogenous retroviruses with the same PBS. Thus, our study sheds light on a strategy for searching for host restriction factors targeting retroviruses.

Structure and function of retroviral integrase.

A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell chromosome. Integrase is the viral enzyme responsible for the catalytic steps involved in this process, and integrase strand transfer inhibitors are widely used to treat people living with HIV. Over the past decade, a series of X-ray crystallography and cryogenic electron microscopy studies have revealed the structural basis of retroviral DNA integration. A variable number of integrase molecules congregate on viral DNA ends to assemble a conserved intasome core machine that facilitates integration. The structures additionally informed on the modes of integrase inhibitor action and the means by which HIV acquires drug resistance. Recent years have witnessed the development of allosteric integrase inhibitors, a highly promising class of small molecules that antagonize viral morphogenesis. In this Review, we explore recent insights into the organization and mechanism of the retroviral integration machinery and highlight open questions as well as new directions in the field.

ICTV Virus Taxonomy Profile: Retroviridae 2021.

Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80-100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV.

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5-10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT's activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

Influence of Different Glycoproteins and of the Virion Core on SERINC5 Antiviral Activity.

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.

Discovery and Characterization of Actively Replicating DNA and Retro-Transcribing Viruses in Lower Vertebrate Hosts Based on RNA Sequencing.

In a previous study, a metatranscriptomics survey of RNA viruses in several important lower vertebrate host groups revealed huge viral diversity, transforming the understanding of the evolution of vertebrate-associated RNA virus groups. However, the diversity of the DNA and retro-transcribing viruses in these host groups was left uncharacterized. Given that RNA sequencing is capable of revealing viruses undergoing active transcription and replication, we collected previously generated datasets associated with lower vertebrate hosts, and searched them for DNA and retro-transcribing viruses. Our results revealed the complete genome, or "core gene sets", of 18 vertebrate-associated DNA and retro-transcribing viruses in cartilaginous fishes, ray-finned fishes, and amphibians, many of which had high abundance levels, and some of which showed systemic infections in multiple organs, suggesting active transcription or acute infection within the host. Furthermore, these new findings recharacterized the evolutionary history in the families Hepadnaviridae, Papillomaviridae, and Alloherpesviridae, confirming long-term virus-host codivergence relationships for these virus groups. Collectively, our results revealed reliable and sufficient information within metatranscriptomics sequencing to characterize not only RNA viruses, but also DNA and retro-transcribing viruses, and therefore established a key methodology that will help us to understand the composition and evolution of the total "infectome" within a diverse range of vertebrate hosts.

The origin of porcine endogenous retroviruses (PERVs).

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs, and they produce viral particles that are able to infect human cells and therefore pose a special risk for xenotransplantation. In contrast to other pig microorganisms that also pose a risk, such as porcine cytomegalovirus and hepatitis E virus, PERVs cannot be eliminated from pigs by vaccines, antiviral drugs, early weaning, or embryo transfer. Since PERVs are relevant for xenotransplantation, their biology and origin are of great interest. Recent studies have shown that PERVs are the result of a transspecies transmission of precursor retroviruses from different animals and further evolution in the pig genome. PERVs acquired different long terminal repeats (LTRs), and recombination took place. In parallel, it has been shown that the activity of the LTRs and recombination in the envelope are important for the transmissibility and pathogenesis of PERVs. Transspecies transmission of retroviruses is common, a well-known example being the transmission of precursor retroviruses from non-human primates to humans, resulting in human immunodeficiency virus (HIV). Here, recent findings concerning the origin of PERVs, their LTRs, and recombination events that occurred during evolution are reviewed and compared with other findings regarding transspecies transmission of retroviruses.

Novel viruses detected in bats in the Republic of Korea.

Bats are natural reservoirs for potential zoonotic viruses. In this study, next-generation sequencing was performed to obtain entire genome sequences of picornavirus from a picornavirus-positive bat feces sample (16BF77) and to explore novel viruses in a pooled bat sample (16BP) from samples collected in South Korea, 2016. Fourteen mammalian viral sequences were identified from 16BF77 and 29 from 16BP, and verified by RT-PCR. The most abundant virus in 16BF77 was picornavirus. Highly variable picornavirus sequences encoding 3Dpol were classified into genera Kobuvirus, Shanbavirus, and an unassigned group within the family Picornaviridae. Amino acid differences between these partial 3Dpol sequences were ≥ 65.7%. Results showed that one bat was co-infected by picornaviruses of more than two genera. Retrovirus, coronavirus, and rotavirus A sequences also were found in the BP sample. The retrovirus and coronavirus genomes were identified in nine and eight bats, respectively. Korean bat retroviruses and coronavirus demonstrated strong genetic relationships with a Chinese bat retrovirus (RfRV) and coronavirus (HKU5-1), respectively. A co-infection was identified in one bat with a retrovirus and a coronavirus. Our results indicate that Korean bats were multiply infected by several mammal viruses.

A Novel Retrovirus (Gunnison's Prairie Dog Retrovirus) Associated With Thymic Lymphoma in Gunnison's Prairie Dogs in Colorado, USA.

As part of research and wildlife disease surveillance efforts, we performed necropsy examinations of 125 free-ranging (n = 114) and captive (n = 11) prairie dogs in Colorado from 2009 to 2017. From these cases, we identified three cases of thymic lymphoma in free-ranging Gunnison's prairie dogs (Cynomys gunnisoni), and we identified a novel retroviral sequence associated with these tumors. The viral sequence is 7700 nucleotides in length and exhibits a genetic organization that is consistent with the characteristics of a type D betaretrovirus. The proposed name of this virus is Gunnison's prairie dog retrovirus (GPDRV). We screened all 125 prairie dogs for the presence of GPDRV using PCR with envelope-specific primers and DNA extracted from spleen samples. Samples were from Gunnison's prairie dogs (n = 59), black-tailed prairie dogs (Cynomys ludovicianus) (n = 40), and white-tailed prairie dogs (Cynomys leucurus) (n = 26). We identified GPDRV in a total of 7/125 (5.6%) samples including all three of the prairie dogs with thymic lymphoma, as well as spleen from an additional four Gunnison's prairie dogs with no tumors recognized at necropsy. None of the GPDRV-negative Gunnison's prairie dogs had thymic lymphomas. We also identified a related, apparently endogenous retroviral sequence in all prairie dog samples. These results suggest that GPDRV infection may lead to development of thymic lymphoma in Gunnison's prairie dogs.

Retroviruses in the pathogenesis of systemic lupus erythematosus: Are they potential therapeutic targets?

The pathogenesis of systemic lupus erythematosus (SLE) is characterised by the hyper-activation of immunologic pathways related to the antiviral response. Exogenous and endogenous retroviruses, by integrating their DNA templates in the host cell genome, may epigenetically control the transcription of genes involved in the immune response. Furthermore, their nucleic acids or neo-synthesized proteins could stimulate the sensor molecules placed upstream the inflammatory cascade. Exogenous retroviruses, like human immunodeficiency virus, have been associated to SLE-like manifestations or to a fair SLE diagnosis. In addition, there is some evidence confirming a pathogenic role of human endogenous retroviruses in SLE. In line with these data, the use of antiretroviral agents could represent an attractive opportunity in the future therapeutic algorithms of this disease, but studies are still missing.

The Current View of Retroviruses as Seen from the Shoulders of a Giant.

It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].

New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic ß cell lines.

BACKGROUND: Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing ß cells. However, few studies have used vectors derived from « simple ¼ retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into ß cells. RESULTS: We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian ß cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in ß over non-ß cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human ß cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human ß cell lines. CONCLUSION: Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian ß cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of ß cell function and monitor changes in their differentiation status.

Human endogenous retrovirus-K (HML-2): a comprehensive review.

The human genome contains a large number of retroviral elements acquired over the process of evolution, some of which are specific to primates. However, as many of these are defective or silenced through epigenetic changes, they were historically considered "junk DNA" and their potential role in human physiology or pathological circumstances have been poorly studied. The most recently acquired, human endogenous retrovirus-K (HERV-K), has multiple copies in the human genome and some of them have complete open reading frames that are transcribed and translated, especially in early embryogenesis. Phylogenetically, HERV-K is considered a supergroup of viruses. One of the subtypes, termed HML-2, seems to be the most active and hence, it is the best studied. Aberrant expression of HML-2 in adult tissues has been associated with certain types of cancer and with neurodegenerative diseases. This review discusses the discovery of these viruses, their classification, structure, regulation and potential for replication, physiological roles, and their involvement in disease pathogenesis. Finally, it presents different therapeutic approaches being considered to target these viruses.

Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

Genomic fossils reveal adaptation of non-autonomous pararetroviruses driven by concerted evolution of noncoding regulatory sequences.

The interplay of different virus species in a host cell after infection can affect the adaptation of each virus. Endogenous viral elements, such as endogenous pararetroviruses (PRVs), have arisen from vertical inheritance of viral sequences integrated into host germline genomes. As viral genomic fossils, these sequences can thus serve as valuable paleogenomic data to study the long-term evolutionary dynamics of virus-virus interactions, but they have rarely been applied for this purpose. All extant PRVs have been considered autonomous species in their parasitic life cycle in host cells. Here, we provide evidence for multiple non-autonomous PRV species with structural defects in viral activity that have frequently infected ancient grass hosts and adapted through interplay between viruses. Our paleogenomic analyses using endogenous PRVs in grass genomes revealed that these non-autonomous PRV species have participated in interplay with autonomous PRVs in a possible commensal partnership, or, alternatively, with one another in a possible mutualistic partnership. These partnerships, which have been established by the sharing of noncoding regulatory sequences (NRSs) in intergenic regions between two partner viruses, have been further maintained and altered by the sequence homogenization of NRSs between partners. Strikingly, we found that frequent region-specific recombination, rather than mutation selection, is the main causative mechanism of NRS homogenization. Our results, obtained from ancient DNA records of viruses, suggest that adaptation of PRVs has occurred by concerted evolution of NRSs between different virus species in the same host. Our findings further imply that evaluation of within-host NRS interactions within and between populations of viral pathogens may be important.

A computational model for predicting integrase catalytic domain of retrovirus.

Integrase catalytic domain (ICD) is an essential part in the retrovirus for integration reaction, which enables its newly synthesized DNA to be incorporated into the DNA of infected cells. Owing to the crucial role of ICD for the retroviral replication and the absence of an equivalent of integrase in host cells, it is comprehensible that ICD is a promising drug target for therapeutic intervention. However, annotated ICDs in UniProtKB database have still been insufficient for a good understanding of their statistical characteristics so far. Accordingly, it is of great importance to put forward a computational ICD model in this work to annotate these domains in the retroviruses. The proposed model then discovered 11,660 new putative ICDs after scanning sequences without ICD annotations. Subsequently in order to provide much confidence in ICD prediction, it was tested under different cross-validation methods, compared with other database search tools, and verified on independent datasets. Furthermore, an evolutionary analysis performed on the annotated ICDs of retroviruses revealed a tight connection between ICD and retroviral classification. All the datasets involved in this paper and the application software tool of this model can be available for free download at https://sourceforge.net/projects/icdtool/files/?source=navbar.

Detection of koala retrovirus subgroup B (KoRV-B) in animals housed at European zoos.

Many koalas carry an endogenous retrovirus, KoRV-A, in their genome. Recently, a second retrovirus, KoRV-B, was detected in koalas in Japanese and U.S. zoos. However, this virus is not endogenous, differs in the receptor binding site of the surface envelope protein, and uses a receptor different from that of KoRV-A. We describe here a KoRV-B found in koalas at zoos in Germany and Belgium that differs slightly from that found in the Los Angeles zoo.

The Discovery of Reverse Transcriptase.

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.