The 15th international dsRNA virus symposium, Porto, Portugal, November 3-7, 2025.

Replication of the positive-strand RNA viruses generates double-stranded RNAs (dsRNAs) that are recognized by host pattern recognition receptors (PRRs) to trigger innate immune responses. Formation of the viral replication complex (RC) has been thought to shield dsRNA from being recognized by innate sensors. To elucidate the RC-mediated evasion of innate recognition, we selected poliovirus (PV) as a model. We first found that RNAs generated during PV replication were potent interferon (IFN) inducers upon transfection, while there was no obvious IFN production detected in PV-replicating cells. PV replication did not interfere with IFN production when IFN agonists were synchronously introduced with the replicating PV RNAs, and in PV-infected cells, IFN agonist-induced IFN production was only moderately impaired but not completely abolished. When PV-infected cells were in situ permeabilized by digitonin, viral dsRNAs were readily detected by an anti-dsRNA antibody and were resistant to RNase III digestion. When digitonin-permeabilized cells were further solubilized by 1 % triton X-100, the dsRNAs of PV became sensitive to RNase III digestion. A co-localization study showed that PV dsRNA did not co-localize with MDA5 in virally infected cells. Given that the PV replication complex is protruding single-membrane and tubular in form, viral replicative dsRNAs are probably shielded by the replication complex or the viral replicase to avoid being accessed by RNase III and MDA5. We propose that the replication complex- or replicase-mediated shielding of dsRNA may act as a means for innate evasion.

Multifaceted roles for lipids in viral infection

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

CVB3 myocarditis can lead to dilated cardiomyopath (DCM). DCM is one of the leading causes of the need for heart transplantation, so it is important to understand the life cycle of CVB3 and its interactions with the host cell. Infection causes rapid death of host cardiomyocytes by altering normal cellular homeostasis for the efficient release of progeny virion. In this chapter, we will examine the impact that CVB3 replication has on host cell biology, from events that take place at receptor ligation to progeny virus release. The primary focus will be on the myriad of signalling pathways that are activated at all stages of virus replication and their downstream effects. We will also discuss some of the extracellular effects of infection as well as immune and matrixmetalloprotease activation. Interactions of host cell proteins with the 5' untranslated region (UTR) are required for translation and replication of CVB3. These interactions do not always benefit the virus since the interactions of a 28-kDa host protein with the 5' UTR are thought to be responsible for inhibitory activity against CVB3. Finally, we will discuss how the elucidation of the different stages of replication has provided the opportunity to develop novel strategies for combating CVB3 infection.

Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

We read with interest the novel strategy described by Drs. Papendorp and van den Berk in the March 27 issue of AIDS [1] of instituting a standard HAART regimen (tenofovir, emtricitabine, and efavirenz) plus raltegravir to reduce HIV-1 viral load rapidly in order to decrease peri-operative infectious risk. While raltegravir-containing HAART affords a more rapid viral load decline compared to other antiretroviral regimens [2], there remains a period of several weeks before virions are cleared from the plasma. This may represent an unacceptably long time to wait for urgent surgery. Building upon Drs. Papendorp and van den Berk’s innovative concept of using the pharmacodynamic properties unique to particular classes of antiretrovirals to reduce peri-operative risk, we describe an alternative strategy that theoretically offers the same benefit within a much shorter time period. Raltegravir, the first integrase inhibitor approved for clinical use, blocks incorporation of HIV-1 reverse transcripts into the host cell genome. Infected cells that have already undergone HIV-1 DNA integration at the time raltegravir is initiated will not be blocked from producing infectious virions. The rapid viral load decay seen in patients taking raltegravir may be the result of the blockade of a later stage of viral replication than other currently available antiretroviral drugs [3]. For example, it is likely that a larger population of infected cells can continue to produce virus in patients starting an efavirenz-based regimen than in patients starting a raltegravir-based regimen. With efavirenz initiation, all infected cells that have undergone reverse transcription will continue to produce virus, whereas with raltegravir only cells that have undergone viral integration will produce virus. This is likely the basis of the more rapid decay in plasma HIV-1 RNA levels in patients starting raltegravir [3]. However, with respect to occupational exposure, the critical issue is not the absolute level of plasma virus, but rather the level of infectious virus. In theory, virions produced by previously infected cells in a patient starting an integrase inhibitor will be fully infectious if transferred to another host, because integrase inhibitors act only on the integrase-viral DNA complex in newly infected cells and not on the free enzyme in the virion [4]. The situation is different in patients starting protease inhibitors. These drugs inhibit the enzyme responsible for the cleavage of immature viral proteins within the virion during and after budding from the host cell. Previously infected cells at all stages of replication will continue to produce virions when a protease inhibitor is initiated [3]. For this reason, the viral load decay on protease inhibitor regimens is not as rapid as that observed with raltegravir. However, all virions produced in the presence of therapeutic concentrations of a protease inhibitor are immature and incapable of infecting a new target. Thus, within hours of initiating a protease inhibitor-based regimen, patients will have a falling viral load composed of immature, non-infectious virions that likely represent a minimal risk for blood borne viral transmission. In the case of protease inhibitors with a slow off rate such as darunavir [5], enzyme inhibition may persist following occupational transmission of virus particles to a recipient for longer than the virion lifetime, affording complete protection. If the drug does dissociate, the virion would still need to complete the maturation process to become infectious. The non-nucleoside reverse transcriptase inhibitors potentially represent an intermediate case. In principle, they can bind to the reverse transcriptase molecules in virions, rendering them non-infectious. However, as soon as the inhibitor dissociates, reverse transcription could continue. Thus in order to minimize the probability of occupational transmission, slowly dissociating protease inhibitors offer the most rapid way to reduce infectious virus in the plasma. A potential concern of the drug regimen described by Papendorp and van den Berk is the relatively low barrier to resistance of both efavirenz and raltegravir [6], which may present a risk to the patient long after surgery. This risk is compounded by initiating HAART on an urgent basis in a setting radically different from the outpatient HIV clinic, where a support structure is ideally in place to maximize long-term positive patient outcomes. Protease inhibitor-based regimens have a significantly higher barrier to resistance than efavirenz or raltegravir, and for this reason can often be given with confidence in settings when the presence of drug-resistance mutations cannot be fully assessed. In summary, both the mechanism of action and the superior resistance profile of protease inhibitor-based HAART argue for its use in this specific setting. In regard to blood borne transmission risk, the quality as well of the quantity of virus should be considered.

The coronavirus nonstructural proteins (nsp's) derived from the replicase polyproteins collectively constitute the viral replication complexes, which are anchored to double-membrane vesicles. Little is known about the biogenesis of these complexes, the membrane anchoring of which is probably mediated by nsp3, nsp4, and nsp6, as they contain several putative transmembrane domains. As a first step to getting more insight into the formation of the coronavirus replication complex, the membrane topology, processing, and subcellular localization of nsp4 of the mouse hepatitis virus (MHV) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were elucidated in this study. Both nsp4 proteins became N glycosylated, while their amino and carboxy termini were localized to the cytoplasm. These observations imply nsp4 to assemble in the membrane as a tetraspanning transmembrane protein with a Nendo/Cendo topology. The amino terminus of SARS-CoV nsp4, but not that of MHV nsp4, was shown to be (partially) processed by signal peptidase. nsp4 localized to the endoplasmic reticulum (ER) when expressed alone but was recruited to the replication complexes in infected cells. nsp4 present in these complexes did not colocalize with markers of the ER or Golgi apparatus, while the susceptibility of its sugars to endoglycosidase H indicated that the protein had also not traveled trough the latter compartment. The important role of the early secretory pathway in formation of the replication complexes was also demonstrated by the inhibition of coronaviral replication when the ER export machinery was blocked by use of the kinase inhibitor H89 or by expression of a mutant, Sar1[H79G].

Double‐stranded (ds) RNA viruses face two major challenges when infecting a cell: the cellular replicative machinery does not operate on dsRNA genomes, and dsRNA provokes a strong apoptotic response. To overcome these problems, dsRNA viruses conceal their genomes from the host cell in an enclosed icosahedral viral core, but then need to carry with them all the necessary enzymatic requirements for replication and transcription. dsRNA virus genomes are often segmented (up to 12 segments). Whereas the transcription and capping reactions have been well documented (see, for example, Luongo et al ., 2000; Reinisch et al ., 2000), the mechanism used for genome packaging, and the site for minus strand synthesis, have not been well defined. A paper by Diprose et al . (2001) now sheds new light on the trafficking of the precursors and transcription products of an active dsRNA virus core. These cores are symmetrical (icosahedral) particles with 2‐, 3‐ and 5‐fold axes of symmetry (Figure 1; Grimes et al ., 1998). Figure 1. A schematic illustration of a dsRNA virus genome replication cycle. The infection process brings into the cell the core containing the dsRNA genome segments ( a ). Upon uncoating, the particle is activated to synthesize single‐stranded (ss) RNA copies from each genome segment. These exit through machinery occupying the icosahedral particle vertices ( b ). The ssRNA segments are of positive polarity and are utilized as mRNAs ( c ), leading to the synthesis of proteins that …

Many virus infections elicit vigorous host immune responses, both innate and acquired. The immune responses are frequently successful in controlling and then clearing the virus, using both cellular effectors such as natural killer (NK) cells and cytolytic T lymphocytes and soluble factors such as interferons (IFNs). However, some immune responses lead to pathologic changes or are unable to prevent the pathogen’s growth. This review will not be devoted to the different strategies viruses have taken to promote their transmission or survival but rather to one aspect of the innate immune response to infection: the role of nitric oxide (NO) in the antiviral repertoire. Recently, data from many laboratories, using both RNA and DNA viruses in experimental systems, have implicated a role for NO in the immune response. The data do not indicate a magic bullet for all systems but suggest that NO may inhibit an early stage in viral replication and thus prevent viral spread, promoting viral clearance and recovery of the host. The earliest host responses to viral infections are nonspecific and involve the induction of cytokines, among them, IFNs and tumor necrosis factor alpha (TNF-α). Gamma IFN (IFN-γ) and TNF-α have both been shown to be active in many cell types and induce cascades of downstream mediators (reviewed in references 25, 34, and 41). Others have found that NO synthase type 2 (NOS-2, iNOS) is an IFN-γ-inducible protein in macrophages, requiring IRF-1 as a transcription factor (12, 17). We have observed that the isoform expressed in neurons, NOS-1, is IFN-γ, TNF-α, and interleukin-12 (IL-12) inducible (20). Thus, NOS falls into the category of IFN-inducible proteins, activated during innate immune responses. NO is produced by the enzymatic modification of l-arginine to l-citrulline and requires many cofactors, including tetrahydrobiopterine, calmodulin, NADPH, and O2. NO rapidly reacts with proteins or with H2O2 to form ONOO−, peroxynitrite, which is highly toxic (Fig. ​(Fig.1).1). NO also readily binds heme proteins, including Hb and its own enzyme. FIG. 1 Reaction of NO with proteins or H2O2 to form ONOO−. This review will not include a great deal of detail about the biochemistry, pharmacology, and molecular biology of NOSs as there are many excellent review articles available (7, 10, 31, 41). However, to show the range of processes in which NOSs are involved, we will illustrate a few. NO was initially described as a physiological mediator of endothelial cell relaxation, an important role in hypotension (35, 42). An extension of this role is in penile erection (8). NO is central to long-term potentiation in neurons (32) and to activity in the biological clock, the suprachiasmic nucleus (13). There are three well-characterized isoforms of NOS, termed NOS-1, NOS-2, and NOS-3 (Table ​(Table11). TABLE 1 Isoforms of NOS Immunologically, NOS activity, NOS-immunoreactive proteins, and mRNA have been found in autoimmune diseases, such as multiple sclerosis, associated with demyelinating lesions (11) and arthritic joints (40) and are thought to contribute to disease pathogenesis. NOS is frequently observed to be induced during the immune response (5). In contrast, in many intracellular bacterial and parasitic infectious diseases, NOS activity has been observed to be essential in eliminating pathogens such as Plasmodium falciparum (4). In the last 5 years, dozens of articles have been published which show some association between NO and viral infections both in vivo and in vitro. There have been three basic experimental strategies used to determine if NO functionally inhibits viral replication: (i) using NO donors such as sodium nitroprusside (SNP), S-nitroso-l-acetylpenicillamine (SNAP), or 3-morpholino-sydononimine (SIN-1) in vitro; (ii) using analogs of the substrate l-arginine, such as l-NMA, l-NAME, 7-nitroindazole, to inhibit enzyme activity in vitro or in vivo; (iii) infecting host strains of mice which are homozygously deficient (knockouts) in one of the NOS isoforms. Table ​Table22 summarizes many of the findings in RNA and DNA viral systems. TABLE 2 Summary of published findings on the effect(s) of NO on viral infection In vitro, for most (but not all) viruses studied, prior activation of the cell to have enzyme activity before infection is associated with inhibition of viral replication. This has been accomplished by providing NO donors, by coculture with activated macrophages as a source of diffusing NO, or by directly activating NOS in cells with cytokines or through other cell surface receptors (e.g., the glutamate receptor, NMDA-R). This includes both DNA and RNA viruses, enveloped and encapsidated: all picornaviruses tested (20, 27, 28, 33, 39, 45), Japanese encephalitis virus (JEV) (26), mouse hepatitis virus (MHV) (24), vesicular stomatitis virus (VSV) (20), Friend murine leukemia virus (MuLV) (3), herpes simplex virus type 1 (HSV-1) (18, 20), vaccinia virus (16, 18, 30), and ectromelia virus (18). There are several exceptions, some tested with positive controls of IFN-mediated viral inhibition (41), including influenza virus (20), Sindbis virus (20, 44), and tick-borne encephalitis virus (23) (note that another member of the Flaviviridae, JEV, is sensitive [26]). Thus, NO is not a magic bullet in vitro; however, it is very potent for many different viruses. The mechanism of inhibition of viral replication in vitro is actively under investigation in many laboratories. It may be that there will be several different pathways involved, especially given the diversity of virus families which are sensitive or resistant. For coxsackievirus type B3 (CVB3) and JEV, RNA synthesis and protein synthesis are inhibited (26, 45). For VSV, very early protein synthesis is inhibited and the viral structural proteins are nitrosylated (40a). For vaccinia virus, late viral protein synthesis and DNA replication are inhibited (16, 30). In some cases, NOS-2 is detectable in tissues from infected animals and may be attributable to activation of macrophages and microglia by IFN-γ or TNF-α. This has been associated with tissue pathology in several systems: CVB3 (28), borna virus (2, 22), MHV demyelination (14), human immunodeficiency virus (HIV) gp120 neuropathology (36), and HSV-1 pneumonia (1). In vivo, the data on inhibition of viral replication tend to agree with the in vitro findings. That is, when a virus is very sensitive to NO in tissue culture, treatment of infected hosts with an inhibitor of the enzyme is associated with increased viral replication. This was found for VSV (20), Friend MuLV (3), HSV-1 (1), and ectromelia virus (18). An exception was observed with Sindbis virus, since two laboratories found that there was no in vitro sensitivity to NO (20, 44) but mice treated with an inhibitor succumbed more readily during central nervous system (CNS) infection (44); however, this drug was provided in drinking water, which may not have been palatable to sick mice. Exceptions to the correlation were observed for vaccinia virus and for MHV infection of the CNS, for which there was no effect on disease when mice were treated with an inhibitor of NOS (24, 37). In the murine cytomegalovirus (MCMV) system, treatment of mice with l-NMA suggested that NO played a more important role in controlling viral replication in the livers of mice, but NK cells were essential in the spleen for eliminating MCMV (43). Although there are knockout mice for each of the three isoforms of NOS, the experiments with two of the systems have not yet been published. We have found that CNS infection of mice with VSV, which replicates in neurons, requires NOS-1 for clearance of infection, recovery, and survival (20a). NOS-3-deficient mice resembled wild-type mice in their responses to the infection (20a). Lowenstein’s work with NOS-2-deficient mice indicates that CVB3 replicates to higher titers in knockout mice (28a). What does this all mean? NO frequently is an important mediator in intracellular inhibition of viral replication, which results in lower viral yields and more efficient host clearance of the infection, hence recovery. NO is not the only intracellular inhibitor, because many of the IFN-inducible proteins block viral pathways (41). There is no clear-cut way of predicting if NO will have a role in viral clearance or pathogenesis. DNA and RNA viruses are both sensitive or resistant. There are many pathogens which are not inhibited by NO; however, NO may also contribute to tissue damage, especially if substantial numbers of macrophages are activated, producing large quantities of NO, as in Borna disease (2, 22) or HSV-1 pneumonia (1). Since there are many enzyme inhibitors available, those diseases in which NOS-2 activity is detrimental may benefit from enzyme antagonism. Host organ tropism also does not predict the selectivity of this response. However, in the case of viral encephalitis due to infection with picornaviruses, rhabdoviruses, HSV-1, or JEV, for instance, activation of NOS-1 may be lifesaving.

The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

Since the appearance of the first cases of infection in 1981 in New York and San Francisco by a virus unknown at the time and later identified in 1983 as the human immunodeficiency virus (HIV), which causes AIDS and is responsible for some 42 million deaths and 88 million infections since the beginning of the pandemic, fundamental discoveries have been made that have led to great advances in HIV control. These discoveries range from methods of rapid detection of the virus, basic knowledge of the structure and function of viral proteins, mechanisms of cell penetration, receptors, interaction with the host, transport and genomic integration, stability, pathology, to the development of control procedures, especially antivirals. For years, HIV infection was synonymous with death, until the scientific community and the pharmaceutical sector developed antiretrovirals. There are currently multiple specific compounds directed against different stages in viral replication, such as reverse transcriptase inhibitors, nucleoside analogues and non-analogues, protease inhibitors, inhibitors of fusion and penetration of the virus, antagonists of co-receptors on the surface of lymphocytes, integrase blockers, inhibitors of the fusion of the virus to the cell membrane, capsid inhibitors, pharmacokinetic enhancers. These drugs are combined, using at least two drugs in a single pharmaceutical form with two different classes of action for oral treatment and also in long-acting intravenous administration. In this way, it has been possible to turn a fatal infection into a chronic one, with a very high long-term survival rate.

Subcellular localization of the MNV-1 ORF1 proteins and their potential roles in the formation of the MNV-1 replication complex

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

NS4A and NS4B proteins from dengue virus: Membranotropic regions

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ~1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.