Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, RNA1 (replicase gene) and RNA2 (coat protein gene), which are encapsidated into a single virion. Expression of coat protein in Sf21 cells using a baculovirus vector results in formation of virus-like particles (VLPs) whose capsids are structurally indistinguishable from native virions. However, RNA packaging is not specific for RNA2, the coat protein message. Using ribonuclease protection assays, we showed that the fraction of RNA2 in VLPs is 19% relative to the amount present in a population of native virions. To investigate possible reasons for the reduced level of RNA2, we generated two new baculovirus vectors, AcR1delta and AcR2delta, expressing the replicase gene and the coat protein gene, respectively. The inserted genes carried the self-cleaving hepatitis delta ribozyme sequence at the 3' end to allow for synthesis of RNA1 and RNA2 transcripts with authentic 3' ends. Infection of Sf21 cells with AcR2delta yielded VLPs that contained 66% RNA2 relative to native virions. Coinfection of Sf21 cells with AcR1delta and AcR2delta launched self-directed FHV replication and resulted in formation of particles most of which contained RNA1 and RNA2. However, a small fraction of particles containing cellular RNA was detected as well. The latter particles could be eliminated by infecting Sf21 cells with AcR1delta followed by transfection with in vitro synthesized transcripts of RNA2. We have further utilized this system to show that two coat protein deletion mutants with distinct RNA packaging defects form mosaic virus capsids but do not complement each other to rescue specific packaging of FHV RNAs.

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetylaziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.A ntiviral agents usually attack the viral life cycle by inhibiting intracellular expression of viral enzymes or by interfering with extracellular steps such as interaction of the virus with the cellular receptor (1-5). Viral protease and replicase inhibitors are highly specific, but their efficacy can be significantly reduced by the emergence of viral mutants. A more general approach to disarming viruses is through chemical modification of the virus particles, such as with N-acetyl-aziridine (Fig. 1), as used in the production of killed-virus vaccines (6, 7). Here we report how alkylating agents inactivate viruses, and we introduce a versatile molecular design for viral inactivants.Two possible mechanisms exist for viral inactivation with alkylating agents. One mechanism involves the modification of proteins, which would cause inhibition of viral cell entry or the release of the genome. The second mechanism allows the alkylating reagents direct access to the viral genome through a mobile protein capsid (8-12). The recent findings (8-11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation. Focusing on this latter idea, the ability of small alkylating agents to react with either the capsid or encapsidated nucleic acid was investigated initially by using flock house virus (FHV), an RNA-containing model virus used in previous studies (8, 9).The alkylating agent N-acetyl-aziridine is a virus inactivant that has been used in vaccine preparation since the 1950s, yet no direct evidence for its mechanism of inactivation has been determined, making it a suitable starting point for this investigation. The chemistry of aziridines is dominated by ring strain, thus leading to enhanced reactivity in reactions where the strain is relieved. The tendency of aziridines to undergo ring-opening reactions with nucleophiles such as the nitrogen atoms in adenine and guanine make them natural alkylating agents of nucleotides. MethodsCompounds. The synthesis of N-acetyl-aziridine was ...

Assembly of many RNA viruses entails the encapsidation of multiple genome segments into a single virion, and underlying mechanisms for this process are still poorly understood. In the case of the nodavirus Flock House virus (FHV), a bipartite positive-strand RNA genome consisting of RNA1 and RNA2 is copackaged into progeny virions. In this study, we investigated whether the specific packaging of FHV RNA is dependent on an arginine-rich motif (ARM) located in the N terminus of the coat protein. Our results demonstrate that the replacement of all arginine residues within this motif with alanines rendered the resultant coat protein unable to package RNA1, suggesting that the ARM represents an important determinant for the encapsidation of this genome segment. In contrast, replacement of all arginines with lysines had no effect on RNA1 packaging. Interestingly, confocal microscopic analysis demonstrated that the RNA1 packaging-deficient mutant did not localize to mitochondrial sites of FHV RNA replication as efficiently as wild-type coat protein. In addition, gain-of-function analyses showed that the ARM by itself was sufficient to target green fluorescent protein to RNA replication sites. These data suggest that the packaging of RNA1 is dependent on trafficking of coat protein to mitochondria, the presumed site of FHV assembly, and that this trafficking requires a high density of positive charge in the N terminus. Our results are compatible with a model in which recognition of RNA1 and RNA2 for encapsidation occurs sequentially and in distinct cellular microenvironments.

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.

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