Cytomegalovirus Dismantles Cellular Genome Silencing Defense via Viral Protein UL35

Human cytomegalovirus, a widespread herpesvirus that establishes lifelong infections in most of the world’s population, has long been known for its elaborate arsenal of immune-evasion strategies. A new study published in PLOS Pathogens has now revealed a previously unappreciated layer of this molecular arms race, showing that the virus actively dismantles a cellular defense system that silences viral genomes shortly after they enter the nucleus. The research, led by Francesco Montagner, Myriam Scherer, and Thomas Stamminger together with colleagues including Melanie Brinkmann, identifies the viral tegument protein UL35 as a dedicated antagonist of a host factor known as SMC5/6 localization factor 2, or SLF2, and demonstrates that this antagonism is essential for the efficient launch of viral gene expression during the earliest hours of infection.

The cellular defense in question centers on the structural maintenance of chromosomes complex 5/6, a multi-subunit assembly better known for its roles in chromosome biology, including DNA repair and the organization of chromosomal architecture. In recent years, several studies have established that SMC5/6 is not merely a housekeeping complex but also a potent antiviral restriction factor. When episomal DNA viruses, whose genomes persist as circular DNA molecules separate from host chromosomes, attempt to establish infection, SMC5/6 can be recruited to these foreign genomes and impose a repressive chromatin state on them, effectively locking the viral DNA into a transcriptionally silent configuration. Hepatitis B virus, papillomaviruses, and other DNA viruses have all been shown to contend with this restriction, and each has evolved countermeasures, underscoring how fundamental this defense is to the outcome of infection.

A critical insight from prior work is that SMC5/6-mediated silencing of extrachromosomal DNA depends on the physical relocation of the complex to promyelocytic leukemia nuclear bodies, membrane-less nuclear structures long associated with antiviral activity. This recruitment is mediated by SLF2, a protein that acts as a dedicated localization factor, tethering SMC5/6 to these nuclear compartments. Because promyelocytic leukemia nuclear bodies are widely regarded as sites of intrinsic antiviral defense, their integrity is a recurring target of viral attack. Many viruses, including human cytomegalovirus, actively disrupt these structures during infection, dispersing their constituent proteins throughout the nucleoplasm. Given this established interplay, the authors of the new study asked a direct question: what happens to the SMC5/6 complex itself when human cytomegalovirus enters a cell and begins dismantling the very compartments where the complex exerts its antiviral effect?

The answer, arrived at through careful time-course experiments at the onset of infection, was unexpected in one respect and revealing in another. The researchers found that the core components of the SMC5/6 complex were not downregulated by the virus; their abundance remained essentially unchanged as infection proceeded. Instead, the team observed a distinct and reproducible decrease in the protein levels of SLF2, and this loss correlated with the complete dispersal of the SMC5/6 complex away from promyelocytic leukemia nuclear bodies. In other words, the virus did not need to eliminate the restriction complex itself; it needed only to remove the factor responsible for delivering that complex to the compartments where silencing takes place. The strategic logic is elegant: by cutting the tether rather than the payload, the virus prevents the antiviral machinery from ever reaching the incoming viral genomes.

Determining how SLF2 was being eliminated required the researchers to dissect the mechanism of its depletion. Two experimental observations provided important clues. First, the degradation of SLF2 was also observed after infection with ultraviolet-inactivated virus, particles that had been rendered incapable of replicating their genomes or expressing new viral genes. Second, the loss of SLF2 could not be blocked by cycloheximide, a drug that halts cellular protein synthesis and thereby prevents the production of any newly made viral proteins. Together, these findings pointed to a striking conclusion: the factor responsible for SLF2 degradation must be a structural component that is physically packaged into the virus particle and delivered into the cell at the moment of entry, rather than a protein synthesized later in the replicative cycle. Such virion-delivered effectors are a hallmark of herpesvirus biology, allowing these viruses to act against host defenses before their own genes have even begun to be expressed.

To pinpoint the culprit among the virion’s structural proteins, the researchers conducted a targeted screen of candidate tegument proteins, the layer of viral proteins that sits between the viral envelope and the capsid and is released into the cell upon entry. After excluding a requirement for the well-characterized tegument protein pp71, encoded by the UL82 gene, the screen identified UL35 as the protein that is both required and sufficient for the proteasomal degradation of SLF2. The proteasome is the cell’s principal machinery for destroying damaged or unwanted proteins, and viruses routinely hijack it to eliminate host factors that stand in their way. The demonstration that UL35 co-opts this pathway to remove SLF2 places the protein in a growing family of viral effectors that weaponize cellular protein quality control against innate and intrinsic immunity.

Genetic confirmation of UL35’s role came from experiments with a recombinant human cytomegalovirus carrying a stop codon within the UL35 gene region, rendering the protein nonfunctional. In cells infected with this mutant virus, SLF2 protein levels remained stable, and the consequences for the virus were substantial: the mutant displayed a clear defect in the onset of immediate early gene expression, the very first wave of viral transcription that must occur for infection to proceed. This finding connects the molecular mechanism directly to viral fitness, showing that the removal of SLF2 is not a side effect of infection but a functional prerequisite for productive replication. Without UL35, the virus struggles to switch on its genes, precisely the outcome one would expect if the SMC5/6 silencing system were left intact and operational.

Microscopic and biochemical analyses of the mutant infection provided a vivid picture of what happens when the viral antagonist is absent. In cells infected with the UL35-deficient virus, the researchers observed enhanced recruitment of the SMC5/6 complex to promyelocytic leukemia nuclear bodies, and, critically, an overall increase in the association of parental viral genomes, the incoming DNA molecules that have not yet replicated, with these SMC5/6-containing nuclear compartments. The incoming viral genomes, in effect, were being corralled into the very structures where episomal DNA is silenced. This observation provides direct evidence that the SLF2-dependent localization pathway acts on cytomegalovirus genomes during the earliest phase of infection and that UL35 exists, at least in part, to prevent this sequestration from happening.

The final piece of mechanistic confirmation came from a complementary loss-of-function approach. When the researchers depleted SLF2 using small interfering RNAs in cells infected with the UL35-deficient virus, the phenotype was reversed: the accumulation of SMC5/6 at nuclear bodies and the association of viral genomes with those compartments were abolished. This epistasis experiment demonstrates that SLF2 is the relevant host target through which UL35 exerts its effect, and it firmly establishes UL35 as a novel viral antagonist of the SLF2-dependent arm of the SMC5/6 restriction system. The logic of the pathway is now complete: SLF2 delivers SMC5/6 to promyelocytic leukemia nuclear bodies, where the complex can silence incoming episomal viral genomes; UL35, delivered as a structural component of the infecting virion, triggers proteasomal degradation of SLF2 before this localization can occur, thereby sparing the viral genomes from silencing and enabling immediate early gene expression to begin on schedule.

Beyond its immediate mechanistic contribution, the study carries broader implications for virology and antiviral research. It adds human cytomegalovirus to the list of DNA viruses known to counter the SMC5/6 pathway, reinforcing the idea that this complex represents a widely deployed, evolutionarily conserved barrier against episomal DNA. It also highlights the strategic importance of virion-delivered tegument proteins, which allow herpesviruses to neutralize intrinsic immunity within hours of entry, before any adaptive or even transcriptional responses can mount. From a translational perspective, the SLF2-SMC5/6 axis and its viral antagonists offer potential points of intervention: compounds that stabilize SLF2, block UL35 function, or enhance the recruitment of SMC5/6 to viral genomes might tip the balance toward silencing of incoming cytomegalovirus genomes, particularly relevant for immunocompromised patients in whom reactivation and de novo infection remain serious clinical problems. As with other recently discovered antiviral restriction systems, the discovery that a major herpesvirus dedicates a structural protein to dismantling this pathway is itself testimony to how consequential the defense is, and it suggests that further exploration of SMC5/6 biology may yield fresh strategies against a virus that, despite decades of research, still lacks a vaccine and remains a leading cause of congenital disease.

Subject of Research: How human cytomegalovirus protein UL35 antagonizes SMC5/6-mediated silencing of viral genomes by degrading SLF2

Article Title: Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

Article References: Montagner, F., Scherer, M., Stegmann, C., Hahn, F., Ploil, C., Einsiedler, C. S., Brosch, E. K., Maeki, T., Ramani, R., Andrée-Busch, N., Brinkmann, M. M., & Stamminger, T. (2026). Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2. PLOS Pathogens, 22(10), e1014709. https://doi.org/10.1371/journal.ppat.1014709

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014709

Keywords: human cytomegalovirus, SMC5/6, SLF2, UL35, PML nuclear bodies, proteasomal degradation, viral tegument, intrinsic immunity, episomal DNA silencing, immediate early gene expression, herpesvirus, antiviral restriction factors