Viruses as ‘Truffle Hounds’: Molecular Tools for Untangling Brain Cellular Pathology
Authors: Andreas Müller-Schiffmann, Svenja V. Trossbach, Vishwanath R. Lingappa, Carsten Korth
Journal: Trends in Neurosciences (2020)

Neurotropic viruses hijack host-cell proteins and modulate their physiological functions, ultimately leading to changes in proteostasis.These host-cell proteins are often components of multiprotein complexes (MPCs) and some of them overlap with key proteins implicated in neurodegenerative diseases.Modulation of host-cell MPCs is likely to involve the noncanonical 'moonlighting functions' of specific proteins and includes their exploitation for the catalysis of virus assembly and replication.The fast evolutionary cycles of viruses have optimized them to exploit disease-associated weak spots in cellular homeostasis, often in ways unrelated to what can be discovered by the conventional tools of modern drug discovery.We argue that it might be possible to harness these features of viruses to reveal molecular bases for cellular pathology and to identify novel pharmacological targets to treat neurodegenerative diseases. The ability of viruses to evolve several orders of magnitude faster than their host cells has enabled them to exploit host cellular machinery by selectively recruiting multiprotein complexes (MPCs) for their catalyzed assembly and replication. This hijacking may depend on alternative, 'moonlighting' functions of host proteins that deviate from their canonical functions thereby inducing cellular pathology. Here, we posit that if virus-induced cellular pathology is similar to that of other, unknown (non-viral) causes, the identification and molecular characterization of the host proteins involved in virus-mediated cellular pathology can be leveraged to decipher the non-viral disease-relevant mechanisms. We focus on how virus-induced aberrant proteostasis and protein aggregation resemble the cellular pathology of sporadic neurodegenerative diseases (NDs) and how this can be exploited for drug discovery. The ability of viruses to evolve several orders of magnitude faster than their host cells has enabled them to exploit host cellular machinery by selectively recruiting multiprotein complexes (MPCs) for their catalyzed assembly and replication. This hijacking may depend on alternative, 'moonlighting' functions of host proteins that deviate from their canonical functions thereby inducing cellular pathology. Here, we posit that if virus-induced cellular pathology is similar to that of other, unknown (non-viral) causes, the identification and molecular characterization of the host proteins involved in virus-mediated cellular pathology can be leveraged to decipher the non-viral disease-relevant mechanisms. We focus on how virus-induced aberrant proteostasis and protein aggregation resemble the cellular pathology of sporadic neurodegenerative diseases (NDs) and how this can be exploited for drug discovery. The molecular basis for most chronic brain diseases including NDs remains largely unknown. Although they can be categorized by clinical symptoms and, in many cases, by neuropathological phenotypes, a number of external and internal factors such as common genetic variants, infections, immune activation, and toxic substances converge to cause clinical phenotypes by disturbing fundamental networks of cellular homeostasis in the brain. Rare familial mutations causing clinical phenotypes similar to the sporadic ones, have enabled the delineation of some major molecular pathways, but have not as yet yielded clear approaches to therapeutics for the majority of sporadic cases of NDs. Viruses may provide an orthogonal alternative. In medicine, viruses are mainly perceived as disease-causing agents, whereas in biology their fundamental roles in ecological homeostasis [1.Gregory A.C. et al.Marine DNA viral macro- and microdiversity from pole to pole.Cell. 2019; 177: 1109-1123.e14Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar] and as a source of evolutionary novelty are often emphasized [2.Chuong E.B. et al.Regulatory activities of transposable elements: from conflicts to benefits.Nat. Rev. Genet. 2017; 18: 71-86Crossref PubMed Scopus (402) Google Scholar]. Viruses usurp the replication mechanisms of their host cells and do not maintain their own, autonomous cellular metabolism. While it remains controversial how viruses originated [3.Kajan G.L. et al.Virus–host coevolution with a focus on animal and human DNA viruses.J. Mol. Evol. 2020; 88: 41-56Crossref PubMed Scopus (4) Google Scholar], it is clear that viruses coevolve with their hosts [4.Van Doorslaer K. et al.Unique genome organization of non-mammalian papillomaviruses provides insights into the evolution of viral early proteins.Virus Evol. 2017; 3vex027Crossref PubMed Google Scholar]. Consistent with a history of virus–host cell coevolution, beneficial effects of viruses for adaptation have repeatedly been demonstrated, at both the phylogenetic and the ontogenetic level. Especially retroviruses can be seen as genetic editors that left behind numerous endogenous retroviral transcripts in the host genome, some of them of clear utility to the host. It has been estimated that 5–8% of the human genome comprises the remnants of retroviruses [5.Lander E.S. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (16101) Google Scholar,6.de Koning A.P. et al.Repetitive elements may comprise over two-thirds of the human genome.PLoS Genet. 2011; 7e1002384Crossref PubMed Scopus (577) Google Scholar] and once integrated, retroviral sequences frequently shaped the host for good. One example is Arc (Arg3.1), an important regulator of synaptic plasticity [7.Zhang W.C. et al.Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Google Scholar, 8.Campillos M. et al.Computational characterization of multiple Gag-like human proteins.Trends Genet. 2006; 22: 585-589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9.Beique J.C. et al.Arc-dependent synapse-specific homeostatic plasticity.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 816-821Crossref PubMed Scopus (111) Google Scholar], which is one of more than 100 human proteins that have been domesticated from endogenous retroviral gag genes [10.Johnson W.E. Origins and evolutionary consequences of ancient endogenous retroviruses.Nat. Rev. Microbiol. 2019; 17: 355-370Crossref PubMed Scopus (40) Google Scholar]. Likewise, syncytin, a protein essential for mammalian placenta formation, was derived from a retroviral env gene [11.Mi S. et al.Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis.Nature. 2000; 403: 785-789Crossref PubMed Scopus (1008) Google Scholar]. Transposable elements are involved in embryonal development, pluripotency, and cell differentiation in eukaryotes [2.Chuong E.B. et al.Regulatory activities of transposable elements: from conflicts to benefits.Nat. Rev. Genet. 2017; 18: 71-86Crossref PubMed Scopus (402) Google Scholar] and some mammalian proteins are now under the control of endogenous retroviral long-terminal repeats, such as salivary amylase [12.Ting C.N. et al.Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene.Genes Dev. 1992; 6: 1457-1465Crossref PubMed Google Scholar]. Endogenous retroviral elements have also been strongly implicated in shaping host immune responses [13.Grandi N. Tramontano E. Human endogenous retroviruses are ancient acquired elements still shaping innate immune responses.Front. Immunol. 2018; 9: 2039Crossref PubMed Scopus (40) Google Scholar]. The ultrafast generation time of viruses compared with that of their host cells has accelerated viruses' evolution and facilitated their ability to repurpose host cellular machinery to their ends. The host, in turn, is under selection pressure to counter the virus, but is impeded by the longer generation time. By virtue of their success at this evolutionary arm's race, viruses can be used to identify and interrogate weak links in molecular pathways leading to cellular pathologies in their hosts. These same weak links may become dysfunctional in taxing contexts independent of virus infection, including those involved in the pathophysiology of both genetic and sporadic NDs. Studying the interaction of viruses with their host cells may thus offer a fruitful way to reveal cellular homeostatic feedback loops that are not easily accessible using the standard molecular tools available today. Here we advance the concept that if the cellular pathology of a non-viral disease resembles that of a virus-induced disease, the identification of specific virus-targeted host factors (see Glossary) can be used to decipher common molecular disease pathways. This approach seems particularly useful for the so-called 'sporadic' forms of disease (i.e., where the cause is unknown and where no obvious genetic, infectious, or toxic origins can be identified). While in principle such a research and drug discovery strategy can be pursued for most diseases, here we focus on the cellular pathology of NDs that can be related to clear cellular phenotypes of aberrant proteostasis and protein aggregation. It is instructive to look into cancer research, where the strategy to analyze virus–host interactions to identify causes of uncontrolled cell growth and invasion has a much longer tradition and has led to key insights, and we refer readers to Box 1 for a brief discussion of this area.Box 1Lessons Learned from Cancer-Causing VirusesIn 2018, about 2.2 million cancer cases (13% of total cancer cases) had an obvious infectious etiology, with two-thirds owing to direct infection with oncoviruses [93.de Martel C. et al.Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis.Lancet Glob. Health. 2020; 8: e180-e190Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. Since the first discovery of an oncovirus by Peyton Rous in 1911 [33.Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells.J. Exp. Med. 1911; 13: 397-411Crossref PubMed Google Scholar], seven human cancer viruses have now been identified. A hallmark of human oncoviruses is their prolonged, persistent infection in the host that can be lifelong and lead to tumor induction, which has to be viewed as collateral damage from their intracellular presence rather than a consequence of the viral life cycle. Infections with tumor viruses alone are therefore insufficient for cancer development, but add to other, non-infectious cofactors such as genetic mutations or environmental factors, which become enriched during aging and at some tipping point initiate disease. However, the direct implication of viruses in the triggering of cancers has already led to the successful development of two anticancer vaccines, targeting high-risk papillomaviruses and hepatitis B virus [94.Stanley M. Tumour virus vaccines: hepatitis B virus and human papillomavirus.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20160268Crossref PubMed Scopus (18) Google Scholar].Mechanistically, tumor viruses impact cellular proliferation in different ways. Tumor retroviruses either directly express viral oncogenes (v-onc) that are derived from cellular protooncogenes (c-onc) (e.g., c-myc, c-fos, c-erb) or activate the expression of c-onc following integration into the host genome. Small DNA tumor viruses, such as human papillomaviruses (HPVs), polyomaviruses (e.g., SV40), and adenoviruses, encode non-structural multifunctional proteins that target tumor suppressor proteins, such as the moonlighting protein p53 or retinoblastoma protein (pRb), and drive resting cells into the S phase allowing viral replication. Remarkably, oncogenic proteins of different viral families target the same cellular proteins: HPV E6 induces the ubiquitin-mediated degradation of p53, SV40 T-antigen inhibits DNA binding of p53, and adenovirus E1B modulates the transcription-activating function of p53. Similarly, the same viruses affect physiological pRb complex formation by HPV E7, SV40 T-antigen, and adenovirus E1A [95.Butel J.S. Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease.Carcinogenesis. 2000; 21: 405-426Crossref PubMed Google Scholar].Detailed studies of oncoviruses led to the identification of several additional tumor suppressor and oncoproteins that are also involved in non-virally induced tumors and therefore build the basis of our current understanding of the diverse mechanisms involved in general tumor induction [96.Bishop J.M. Molecular themes in oncogenesis.Cell. 1991; 64: 235-248Abstract Full Text PDF PubMed Scopus (1339) Google Scholar], which also gave rise to novel drug targets in cancer therapy. Remarkably, 53 FDA-approved anticancer drugs are directed against tumor-virus-targeted host proteins [97.Guven-Maiorov E. et al.Oncoviruses can drive cancer by rewiring signaling pathways through interface mimicry.Front. Oncol. 2019; 9: 1236Crossref PubMed Scopus (2) Google Scholar]. In 2018, about 2.2 million cancer cases (13% of total cancer cases) had an obvious infectious etiology, with two-thirds owing to direct infection with oncoviruses [93.de Martel C. et al.Global burden of cancer attributable to infections in 2018: a worldwide incidence analysis.Lancet Glob. Health. 2020; 8: e180-e190Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. Since the first discovery of an oncovirus by Peyton Rous in 1911 [33.Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells.J. Exp. Med. 1911; 13: 397-411Crossref PubMed Google Scholar], seven human cancer viruses have now been identified. A hallmark of human oncoviruses is their prolonged, persistent infection in the host that can be lifelong and lead to tumor induction, which has to be viewed as collateral damage from their intracellular presence rather than a consequence of the viral life cycle. Infections with tumor viruses alone are therefore insufficient for cancer development, but add to other, non-infectious cofactors such as genetic mutations or environmental factors, which become enriched during aging and at some tipping point initiate disease. However, the direct implication of viruses in the triggering of cancers has already led to the successful development of two anticancer vaccines, targeting high-risk papillomaviruses and hepatitis B virus [94.Stanley M. Tumour virus vaccines: hepatitis B virus and human papillomavirus.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20160268Crossref PubMed Scopus (18) Google Scholar]. Mechanistically, tumor viruses impact cellular proliferation in different ways. Tumor retroviruses either directly express viral oncogenes (v-onc) that are derived from cellular protooncogenes (c-onc) (e.g., c-myc, c-fos, c-erb) or activate the expression of c-onc following integration into the host genome. Small DNA tumor viruses, such as human papillomaviruses (HPVs), polyomaviruses (e.g., SV40), and adenoviruses, encode non-structural multifunctional proteins that target tumor suppressor proteins, such as the moonlighting protein p53 or retinoblastoma protein (pRb), and drive resting cells into the S phase allowing viral replication. Remarkably, oncogenic proteins of different viral families target the same cellular proteins: HPV E6 induces the ubiquitin-mediated degradation of p53, SV40 T-antigen inhibits DNA binding of p53, and adenovirus E1B modulates the transcription-activating function of p53. Similarly, the same viruses affect physiological pRb complex formation by HPV E7, SV40 T-antigen, and adenovirus E1A [95.Butel J.S. Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease.Carcinogenesis. 2000; 21: 405-426Crossref PubMed Google Scholar]. Detailed studies of oncoviruses led to the identification of several additional tumor suppressor and oncoproteins that are also involved in non-virally induced tumors and therefore build the basis of our current understanding of the diverse mechanisms involved in general tumor induction [96.Bishop J.M. Molecular themes in oncogenesis.Cell. 1991; 64: 235-248Abstract Full Text PDF PubMed Scopus (1339) Google Scholar], which also gave rise to novel drug targets in cancer therapy. Remarkably, 53 FDA-approved anticancer drugs are directed against tumor-virus-targeted host proteins [97.Guven-Maiorov E. et al.Oncoviruses can drive cancer by rewiring signaling pathways through interface mimicry.Front. Oncol. 2019; 9: 1236Crossref PubMed Scopus (2) Google Scholar]. Once a virus is internalized by binding to receptors or by capturing a suitable uptake mechanism, it typically disassembles and starts a highly orchestrated replication process, which involves a massive change in host cellular homeostasis. Depending on the nature of the virus (DNA virus, positive-strand RNA virus, or negative-strand RNA virus), replication of viral nucleic acids (NAs) can take one of two general forms. In the first, replication of viral NA exclusively uses the cell's own NA machinery, as in the case of small DNA viruses such as polyoma- and papillomaviruses. In the second, the viruses provide parts of the replication machinery, which is the case for most RNA viruses and large DNA viruses. For RNA viruses, this is done by the so-called replicase complexes, which are MPCs (Box 2) [14.Choi K.H. Viral polymerases.Adv. Exp. Med. Biol. 2012; 726: 267-304Crossref PubMed Scopus (50) Google Scholar,15.de Castro I.F. et al.Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex.J. Cell Sci. 2017; 130: 260-268Crossref PubMed Scopus (30) Google Scholar], illustrating the point that even the seemingly straightforward process of NA replication is performed by many proteins working together. It is noteworthy that, ultimately, host DNA replication itself is not executed by a single polymerase enzyme but by a complex 'replisome' comprising at least 50 proteins [16.Leman A.R. Noguchi E. The replication fork: understanding the eukaryotic replication machinery and the challenges to genome duplication.Genes (Basel). 2013; 4: 1-32Crossref PubMed Scopus (52) Google Scholar], supporting the notion that MPCs are responsible for viral cellular functions.Box 2Host-Mediated Catalysis of Viral AssemblyA body of literature suggests that viral capsid formation does not occur through spontaneous self-assembly, as had long been believed [98.Bruinsma R.F. et al.Viral self-assembly as a thermodynamic process.Phys. Rev. Lett. 2003; 90: 248101Crossref PubMed Google Scholar]. Instead increasing data support the view that capsid formation is catalyzed by host enzymes [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar,99.Lingappa J.R. et al.A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.J. Cell Biol. 1994; 125: 99-111Crossref PubMed Google Scholar, 100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar, 101.Zimmerman C. et al.Identification of a host protein essential for assembly of immature HIV-1 capsids.Nature. 2002; 415: 88-92Crossref PubMed Scopus (192) Google Scholar]. The evidence for this rests on two different kinds of experiments. First, capsid assembly has been reconstituted in a protein synthesis-dependent manner in cell-free systems and shown to be nucleotide triphosphate hydrolysis-dependent, a telltale sign of enzymatic activity [100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar]. The second is through functional reconstitution of capsid assembly, either by ultracentrifugal fractionation of extracts [100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar] or by affinity chromatography in which small molecules that modulate assembly serve as affinity ligands to fractionate an extract [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar]. In either fractionation approach, the depleted extracts remain capable of capsid protein synthesis, but are no longer capable of assembly. However, assembly can be reconstituted by add-back of the removed fraction. These enzymes of assembly, which have been termed assembly machines, were long overlooked, perhaps because they are transient MPCs, and they could provide a novel molecular basis for homeostasis. Viruses are likely to manipulate signaling pathways [102.Goodwin C.M. et al.Stealing the keys to the kitchen: viral manipulation of the host cell metabolic network.Trends Microbiol. 2015; 23: 789-798Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar] to alter assembly machine composition and action. The resulting aberrant assembly machines favor the needs of the virus over those of host homeostasis. The normal function of those assembly machines are critical points of regulation of host-gene expression. Since, as discussed, viruses in effect run evolution about 50 000× faster than their human hosts, it is unsurprising that they have identified some of our greatest metabolic and biochemical vulnerabilities over deep, evolutionary time – and have coevolved to take advantage of them. If assembly is as important as suggested, assembly machines should be important viral targets, and small molecules targeting allosteric sites that control assembly machine composition and action represent a novel antiviral therapeutic strategy [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar,29.Reed J. et al.Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly.J. Virol. 2020; (Published online November 4, 2020. https://doi.org/10.1128/JVI.00883-20)Crossref PubMed Google Scholar]. It is likely that the same metabolic weak links that viruses take advantage of are the cause of non-viral diseases, in which case assembly modulation would represent an important therapeutic strategy for their treatment [49.Marreiros R. et al.Disruption of cellular proteostasis by H1N1 influenza A virus causes α-synuclein aggregation.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 6741-6751Crossref PubMed Scopus (20) Google Scholar,103.Marreiros R. et al.Viral capsid assembly as a model for protein aggregation diseases: active processes catalyzed by cellular assembly machines comprising novel drug targets.Virus Res. 2015; 207: 155-164Crossref PubMed Scopus (6) Google Scholar]. If we can convert viruses into 'truffle hounds' to identify the high-value drug targets not accessible to conventional proteomics, novel small-molecule therapies as effective at correcting disease as viruses are at causing disease could be identified (see Outstanding Questions). If the above paradigm is correct, two predictions can be made with regard to NDs: (i) there should be a correlation between particular viral families (or, more precisely, their aberrant assembly machines) and particular NDs; and (ii) the small molecules that target aberrant assembly machines should be effective against at least a subset of the non-viral causes of that ND. For both of those predictions, empirical data have recently been reported [57.Sadasivan S. et al.Synergistic effects of influenza and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can be eliminated by the use of influenza therapeutics: experimental evidence for the multi-hit hypothesis.NPJ Parkinsons Dis. 2017; 3: 18Crossref PubMed Google Scholar,77.Li W. et al.Human endogenous retrovirus-K contributes to motor neuron disease.Sci. Transl. Med. 2015; 7307ra153Crossref PubMed Scopus (174) Google Scholar,104.Li Puma D.D. et al.Herpes simplex virus type-1 infection impairs adult hippocampal neurogenesis via amyloid-β protein accumulation.Stem Cells. 2019; 37: 1467-1480Crossref PubMed Scopus (0) Google Scholar]. A body of literature suggests that viral capsid formation does not occur through spontaneous self-assembly, as had long been believed [98.Bruinsma R.F. et al.Viral self-assembly as a thermodynamic process.Phys. Rev. Lett. 2003; 90: 248101Crossref PubMed Google Scholar]. Instead increasing data support the view that capsid formation is catalyzed by host enzymes [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar,99.Lingappa J.R. et al.A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.J. Cell Biol. 1994; 125: 99-111Crossref PubMed Google Scholar, 100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar, 101.Zimmerman C. et al.Identification of a host protein essential for assembly of immature HIV-1 capsids.Nature. 2002; 415: 88-92Crossref PubMed Scopus (192) Google Scholar]. The evidence for this rests on two different kinds of experiments. First, capsid assembly has been reconstituted in a protein synthesis-dependent manner in cell-free systems and shown to be nucleotide triphosphate hydrolysis-dependent, a telltale sign of enzymatic activity [100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar]. The second is through functional reconstitution of capsid assembly, either by ultracentrifugal fractionation of extracts [100.Lingappa J.R. et al.A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.J. Cell Biol. 1997; 136: 567-581Crossref PubMed Scopus (0) Google Scholar] or by affinity chromatography in which small molecules that modulate assembly serve as affinity ligands to fractionate an extract [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar]. In either fractionation approach, the depleted extracts remain capable of capsid protein synthesis, but are no longer capable of assembly. However, assembly can be reconstituted by add-back of the removed fraction. These enzymes of assembly, which have been termed assembly machines, were long overlooked, perhaps because they are transient MPCs, and they could provide a novel molecular basis for homeostasis. Viruses are likely to manipulate signaling pathways [102.Goodwin C.M. et al.Stealing the keys to the kitchen: viral manipulation of the host cell metabolic network.Trends Microbiol. 2015; 23: 789-798Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar] to alter assembly machine composition and action. The resulting aberrant assembly machines favor the needs of the virus over those of host homeostasis. The normal function of those assembly machines are critical points of regulation of host-gene expression. Since, as discussed, viruses in effect run evolution about 50 000× faster than their human hosts, it is unsurprising that they have identified some of our greatest metabolic and biochemical vulnerabilities over deep, evolutionary time – and have coevolved to take advantage of them. If assembly is as important as suggested, assembly machines should be important viral targets, and small molecules targeting allosteric sites that control assembly machine composition and action represent a novel antiviral therapeutic strategy [28.Lingappa U.F. et al.Host–rabies virus protein–protein interactions as druggable antiviral targets.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E861-E868Crossref PubMed Scopus (0) Google Scholar,29.Reed J. et al.Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly.J. Virol. 2020; (Published online November 4, 2020. https://doi.org/10.1128/JVI.00883-20)Crossref PubMed Google Scholar]. It is likely that the same metabolic weak links that viruses take advantage of are the cause of non-viral diseases, in which case assembly modulation would represent an important therapeutic strategy for their treatment [49.Marreiros R. et al.Disruption of cellular proteostasis by H1N1 influenza A virus causes α-synuclein aggregation.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 6741-6751Crossref PubMed Scopus (20) Google Scholar,103.Marreiros R. et al.Viral capsid assembly as a model for protein aggregation diseases: active processes catalyzed by cellular assembly machines comprising novel drug targets.Virus Res. 2015; 207: 155-164Crossref PubMed Scopus (6) Google Scholar]. If we can convert viruses into 'truffle hounds' to identify the high-value drug targets not accessible to conventional proteomics, novel small-molecule therapies as effective at correcting disease as viruses are at causing disease could be identified (see Outstanding Questions). If the above paradigm is correct, two predictions can be made with regard to NDs: (i) there should be a correlation between pa