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Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs, and some of them are able to infect human cells. Therefore, PERVs pose a risk for xenotransplantation, the transplantation of pig cells, tissues, or organ to humans in order to alleviate the shortage of human donor organs. Up to 2021, a huge body of knowledge about PERVs has been accumulated regarding their biology, including replication, recombination, origin, host range, and immunosuppressive properties. Until now, no PERV transmission has been observed in clinical trials transplanting pig islet cells into diabetic humans, in preclinical trials transplanting pig cells and organs into nonhuman primates with remarkable long survival times of the transplant, and in infection experiments with several animal species. Nevertheless, in order to prevent virus transmission to the recipient, numerous strategies have been developed, including selection of PERV-C-free animals, RNA interference, antiviral drugs, vaccination, and genome editing. Furthermore, at present there are no more experimental approaches to evaluate the full risk until we move to the clinic.

Viruses have developed different strategies to escape from immune response. Among these, viral non-coding RNAs are invisible to the immune system and may affect the fate of the host cell. Bovine leukemia virus (BLV) encodes both short (miRNAs) and long (antisense AS1 and AS2) non-coding RNAs. To elucidate the mechanisms associated with BLV non-coding RNAs, we performed phenotypic and transcriptomic analyzes in a reverse genetics system. RNA sequencing of B-lymphocytes revealed that cell proliferation is the most significant mechanism associated with ablation of the viral non-coding RNAs. To assess the biological relevance of this observation, we determined the cell kinetic parameters in vivo using intravenous injection of BrdU and CFSE. Fitting the data to a mathematical model provided the rates of cell proliferation and death. Our data show that deletion of miRNAs correlates with reduced proliferation of the infected cell and lack of pathogenesis.

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Feline leukemia virus (FeLV) is a cosmopolitan gammaretrovirus that causes lifelong infections and fatal diseases, including leukemias, lymphomas, immunodeficiencies, and anemias, in domestic and wild felids. There is currently no definitive treatment for FeLV, and while existing vaccines reduce the prevalence of progressive infections, they neither provide sterilizing immunity nor prevent regressive infections that result in viral reservoirs with the potential for reactivation, transmission, and the development of associated clinical diseases. Previous studies of murine leukemia virus (MuLV) established that host cell epigenetic reader bromodomain and extra-terminal domain (BET) proteins facilitate MuLV replication by promoting proviral integration. Here, we provide evidence that this facilitatory effect of BET proteins extends to FeLV. Treatment with the archetypal BET protein bromodomain inhibitor (+)-JQ1 and FeLV challenge of two phenotypically disparate feline cell lines, 81C fibroblasts and 3201 lymphoma cells, significantly reduced FeLV proviral load, total FeLV DNA load, and p27 capsid protein expression at nonlethal concentrations. Moreover, significant decreases in FeLV proviral integration were documented in 81C and 3201 cells. These findings elucidate the importance of BET proteins for efficient FeLV replication, including proviral integration, and provide a potential target for treating FeLV infections.

Koala retroviruses (KoRV) have been isolated from wild and captive koalas in Australia as well as from koala populations held in zoos in other countries. They are members of the genus Gammaretrovirus, are most closely related to gibbon ape leukemia virus (GaLV), feline leukemia virus (FeLV) and porcine endogenous retrovirus (PERV) and are likely the result of a relatively recent trans-species transmission from rodents or bats. The first KoRV to be isolated, KoRV-A, is widely distributed in the koala population in both integrated endogenous and infectious exogenous forms with evidence from museum specimens older than 150 years, indicating a relatively long engagement with the koala population. More recently, additional subtypes of KoRV that are not endogenized have been identified based on sequence differences and host cell receptor specificity (KoRV-B and KoRV-J). A specific association with fatal lymphoma and leukemia has been recently suggested for KoRV-B. In addition, it has been proposed that the high viral loads found in many animals may lead to immunomodulation resulting in a higher incidence of diseases such as chlamydiosis. Although the molecular basis of this immunomodulation is still unclear, purified KoRV particles and a peptide corresponding to a highly conserved domain in the envelope protein have been shown to modulate cytokine expression in vitro, similar to that induced by other gammaretroviruses. While much is still to be learned, KoRV induced lymphoma/leukemia and opportunistic disease arising as a consequence of immunomodulation are likely to play an important role in the stability of koala populations both in the wild and in captivity.

It has been previously shown that amino acid polymorphisms in reovirus proteins μ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of β-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of μ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Olivomycin A is a highly active antitumor drug that belongs to the family of aureolic acid antibiotics. The antitumor effect of olivomycin A is related to its ability to bind to the DNA minor groove in GC-rich regions as Mg2+-coordinated complexes. Characterization of cellular targets of olivomycin A and its mechanism of action is crucial for the successful application of this antibiotic in clinical practice and development of semi-synthetic derivatives with improved pharmacological properties. Previously, we have shown that minor groove ligands are able to disrupt the key epigenetic process of DNA methylation. In this paper, we have studied the impact of olivomycin A and its improved semi-synthetic analogue N,Ndimethylaminoethylamide of 1′-des-(2,3-dihydroxy-n-butyroyl)-1′-carboxy-olivomycin A (olivamide) on the functioning of de novo DNA methyltransferase Dnmt3a (enzyme that carries out methylation of cytosine residues in the DNA CG-sites in eukaryotic cells) using an in vitro system consisting of the murine Dnmt3a catalytic domain and a 30-mer DNA duplex containing four consecutive GC pairs. We have shown that olivomycin A and olivamide inhibit Dnmt3a with IC50 of 6 ± 1 and 7.1 ± 0.7 μM, respectively. Neither olivomycin A nor olivamide interfered with the formation of the specific enzyme–substrate complex; however, olivomycin A prevented formation of the covalent DNA–Dnmt3a intermediate that is necessary for the methylation reaction to proceed. The inhibitory effects of olivomycin A and olivamide can be explained by the disruption of the enzyme catalytic loop movement through the DNA minor groove (the reaction stage that precedes the covalent bond formation between DNA and the enzyme). The results of this work indicate the epigenetic contribution to the antitumor effect of aureolic acid group antibiotics.