Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features—especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species.

The apparent absence of numerous conserved vertebrate genes from avian genomes has puzzled researchers for over a decade. In recent years, a subset of these genes has been identified; however, their sequences are unusually problematic, often evading detection by standard sequencing technologies. This limitation has hindered detailed investigation of the phenomenon—until recent progress in long read technologies, which are more robust against sequencing biases. This enabled us to classify real gene losses extensively, which strikingly revealed that a large number of the genes residing on so-called dot chromosomes were indeed lost during avian evolution. We demonstrate that dot microchromosomes—small, repeat-dense avian chromosomes—harbor widespread gene attrition, with 29% of ohnologs (duplicates from ancestral genome doublings) eliminated, far exceeding rates on other chromosomes. Moreover, we reveal that genes retained on these dot chromosomes exhibit a previously undescribed form of dynamic genetic instability. This instability, which we term sequence stuttering, is characterized by a massive expansion of short sequences within intronic regions. Intriguingly, in some cases, the expanding sequences appear to originate from neighboring exons. As a result, intron lengths vary extensively among individual chickens, suggesting that these events are evolutionarily recent. Since this phenomenon has not been reported in any other vertebrate species, our findings lay the groundwork for future research into its underlying mechanisms, evolutionary implications, and potential identification of similar loci across vertebrate genomes.

A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Given that the majority of target sequences are not palindromic, the current model explains the symmetry by an overlap of the nonpalindromic motif present on one of the half-sites of the sequences. Here, we show that the implementation of multicomponent mixture models allows for different interpretations consistent with the existence of both palindromic and nonpalindromic submotifs in the sets of integration site sequences. We further show that the weak palindromic motifs result from freely combined site-specific submotifs restricted to only a few positions proximal to the site of integration. The submotifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identify HIV-1-favored palindromic sequences inAlurepeats serving as local hotspots for integration. The application of the novel statistical approach provides deeper insight into the selection of retroviral integration sites and may prove to be a valuable tool in the analysis of any type of DNA motifs.

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.

Retroviruses integrate their genomes into the genomes of infected host cells and form a genetic platform for stable gene expression. Epigenetic silencing can, however, hamper the expression of integrated provirus. As gammaretroviruses (γRVs) preferentially integrate into sites of active promoters and enhancers, the high expression activity of γRVs can be attributed to the integration preference. Long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters for gene expression. Here, we investigate the capacity of different γRV LTRs to drive stable expression inside a non-preferred epigenomic environment using diverse retroviral vectors and CRISPR-Cas9-directed vector knock-in. We demonstrate that different γRV LTRs are either rapidly silenced or long-term active with active proviral population prevailing under normal and retargeted integration. In addition, we show that lamina-associated domains (LADs) can be targeted by CRISPR-Cas9 for vector insertion leading to γRV LTR-driven long-term stable gene expression. Alternatively to established γRV systems, the LTRs of feline leukemia virus and koala retrovirus are capable of driving stable, albeit intensity-diverse, transgene expression in LADs. Altogether, we show that despite the occurrence of rapid silencing events, the majority of γRV LTRs can drive stable expression after retrovirus integration or CRISPR-Cas9-directed knock-in outside of the preferred chromatin landscape.