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Non-enveloped viruses penetrate host membranes to infect cells. A cell-based assay was used to probe the endoplasmic reticulum (ER)-to-cytosol membrane transport of the non-enveloped SV40. We found that, upon ER arrival, SV40 is released into the lumen and undergoes sequential disulfide bond disruptions to reach the cytosol. However, despite these ER-dependent conformational changes, SV40 crosses the ER membrane as a large and intact particle consisting of the VP1 coat, the internal components VP2, VP3, and the genome. This large particle subsequently disassembles in the cytosol. Mutant virus and inhibitor studies demonstrate VP3 and likely the viral genome, as well as cellular proteasome, control ER-to-cytosol transport. Our results identify the sequence of events, as well as virus and host components, that regulate ER membrane penetration. They also suggest that the ER membrane supports passage of a large particle, potentially through either a sizeable protein-conducting channel or the lipid bilayer.

Viral microRNAs MicroRNAs are a large class of noncoding small RNAs, about 22 nucleotides in length, thought to target other genes and regulate their expression. Their regulatory role has been demonstrated in lower organisms: microRNAs are present in mammals including humans, though their role in higher organisms is not clear. Now they have been found to have a function in virus biology. SV40 virus encodes several microRNAs involved in the late stages of infection of monkey cells. These microRNAs maintain viral replicative efficiency by reducing excess antigen production. It has been suggested that inhibitory RNA systems in mammals are there as a defence against viruses, suggesting that what is normally an antiviral defence pathway is being subverted by the virus for its own purposes. MicroRNAs (miRNAs) are small (∼ 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs 1 . miRNAs have also been described in mammalian cells and in infections with Epstein–Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA 2 , the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function 3 . Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an ‘irregularity’ recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane. Non-enveloped viruses such as SV40 are transported from the extracellular space into the host cell nucleus through a pathway involving endocytosis, trafficking to the endoplasmic reticulum (ER) lumen, transport across the ER membrane to the cytoplasm, and subsequent nuclear import. Helenius and colleagues provide insight into how SV40 escapes from the ER by showing that viral proteins interact with components of the host ER-associated degradation machinery (ERAD). These interactions are crucial for translocation of SV40 into the cytoplasm and infectivity.

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, T = 1 VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and T = 1 particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images. Graphic abstract

Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human polyomaviruses. The SV40-GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.

Although the small DNA tumor virus SV40 (simian virus 40) fails to replicate in human cells, understanding how SV40 transforms human and murine cells has and continues to provide important insights into cancer initiation and maintenance. The early region of SV40 encodes two oncoproteins: the large T (LT) and small t (ST) antigens. SV40 LT contributes to murine and human cell transformation in part by inactivating the p53 and retinoblastoma protein tumor suppressor proteins. SV40 ST inhibits the activity of the protein phosphatase 2A (PP2A) family of serine–threonine phosphatases, and this interaction is required for SV40-mediated transformation of human cells. PP2A regulates multiple signaling pathways, suggesting many possible targets important for viral replication and cell transformation. Genetic manipulation of particular PP2A subunits has confirmed a role for specific complexes in transformation, and recent work implicates the perturbation of the phosphatidylinositol 3-kinase/Akt pathway and c-Myc stability in transformation by ST and PP2A. Mutations in PP2A subunits occur at low frequency in human tumors, suggesting that alterations of PP2A signaling play a role in both experimentally induced and spontaneously arising cancers. Unraveling the complexity of PP2A signaling will not only provide further insights into cancer development but may identify novel targets with promise for therapeutic manipulation.

Viruses efficiently transfer and express their genes in host cells and evolve to evade the host's defense responses. These properties render them highly attractive for use as gene delivery vectors in vaccines, gene, and immunotherapies. Among the viruses used as gene delivery vectors, the macaque polyomavirus Simian Virus 40 (SV40) is unique in its capacity to evade intracellular antiviral defense responses upon cell entry. We here describe the unique way by which SV40 particles deliver their genomes in the nucleus of permissive cells and how they prevent presentation of viral antigens to the host's immune system. The non-immunogenicity in its natural host is not only of benefit to the virus but also to us in developing effective SV40 vector-based treatments for today's major human diseases.

Polyomaviruses are a family of small, non-enveloped DNA viruses that can cause severe disease in immunosuppressed individuals. Studies with SV40, a well-studied model polyomavirus, have revealed the role of host proteins in polyomavirus entry and trafficking to the nucleus, in viral transcription and DNA replication, and in cell transformation. In contrast, little is known about host factors or cellular signaling pathways involved in the late steps of productive infection leading to release of progeny polyomaviruses. We previously showed that cytoplasmic vacuolization, a characteristic late cytopathic effect of SV40 infection, depends on the specific interaction between the major viral capsid protein VP1 and its cell surface ganglioside receptor GM1. Here, we show that, late during infection, SV40 activates a signaling cascade in permissive monkey CV-1 cells involving Ras, Rac1, MKK4, and JNK to stimulate SV40-specific cytoplasmic vacuolization and subsequent cell lysis and virus release. Inhibition of individual components of this signaling pathway inhibits vacuolization, lysis, and virus release, even though high-level intracellular virus replication occurs. Identification of this pathway for SV40-induced vacuolization and virus release provides new insights into the late steps of non-enveloped virus infection.