Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies. IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells treated with FDA-approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate nucleic acid packaging, transcription, and translation, but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to the depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential proviral role for polyamines using a forward screen. Passaging CVB3 in polyamine-depleted cells generated a mutation in capsid protein VP3 at residue 234. We show that this mutation confers resistance to polyamine depletion. Through attachment assays, we demonstrate that polyamine depletion limits CVB3 attachment to susceptible cells, which is rescued by incubating virus with polyamines. Furthermore, the capsid mutant rescues this inhibition in polyamine-depleted cells. More divergent viruses also exhibited reduced attachment to polyamine-depleted cells, suggesting that polyamines may facilitate attachment of diverse RNA viruses. These studies inform additional mechanisms of action for polyamine-depleting pharmaceuticals, with implications for potential antiviral therapies. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease. These viruses rely on polyamines, small positively charged molecules, for robust replication, and polyamine depletion limits infection in vitro and in vivo. The mechanisms by which polyamines enhance enteroviral replication are unknown. Here, we describe how Coxsackievirus B3 (CVB3) utilizes polyamines to attach to susceptible cells and initiate infection. Using a forward genetic screen, we identified a mutation in a receptor-binding amino acid that promotes infection of polyamine-depleted cells. These data suggest that pharmacologically inhibiting polyamine biosynthesis may combat virus infection by preventing virus attachment to susceptible cells.

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules are diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively charged molecules involved in cell cycle, translation, and nucleic acid metabolism, among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related molecules associate with purified virions and transmit from cell to cell during infection. Virion-associated spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temperature sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions containing spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.

24Viruses require host cell metabolites to productively infect, and the mechanisms by which 25 viruses usurp these molecules is diverse. One group of cellular metabolites important in virus 26 infection is the polyamines, small positively-charged molecules involved in cell cycle, translation, 27 and nucleic acid synthesis, among other cellular functions. Polyamines also support replication 28 of diverse viruses, and they are important for processes such as transcription, translation, and 29 viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative-sense RNA virus that 30 requires polyamines to produce infectious particles. In polyamine depleted conditions, 31 noninfectious particles are produced that interfere with virus replication and stimulate immune 32 signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity. 33Mastrodomenico et al. 2We show that RVFV replication is facilitated by any of the three biogenic polyamines; however, 34 we specifically find spermidine associated with purified virions. Using a panel of polyamine 35 homologs, we observe that virions can also associate with (R)-3-methylspermidine and 36 norspermidine, though not with other less homologous molecules. Using polyamine reporter 37 cells, we demonstrate that virion-associated polyamines transmit from one infected cell to 38 another. Finally, we find that virions devoid of polyamines are unstable and cannot be 39 supplemented with exogenous polyamines to regain stability or infectivity. These data highlight 40 a unique role for polyamines, and spermidine in particular, in maintaining virus infectivity, a 41 function not previously appreciated. Further, these studies are the first to identify polyamines 42 associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, 43 and this work highlights a new mechanism by which we can inhibit virus replication through 44 FDA-approved polyamine depleting pharmaceuticals. 45 46 the virus continues to present itself in frequent outbreaks 9-11 , infecting hundreds and severely 56 impacting local economies. Thus, the development of improved vaccines or the identification of 57 novel antiviral targets is essential to the treatment and prevention of RVFV. 58 59As obligate intracellular pathogens, viruses rely on their host cells for the building blocks of 60 replication. These building blocks include a variety of metabolites produced by the host cell. 61One set of these metabolites crucial to virus replication is the family of polyamines. Eukaryotic 62 cells synthesize polyamines to support transcription, translation, and cell cycling 12-14 . The 63 biogenic polyamines include putrescine, spermidine, and spermine, which are maintained at 64 millimolar level within cells 15 and readily interconvert within cells 16 . These molecules are carbon 65 chains of increasing length with primary and secondary amine groups. At physiological pH, 85% of polyamines are bound to nucleic acids (primarily RNA), proteins, or lipids t...

From soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit.