Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log₁₀ to 1.88 log₁₀). After 24 h, the reductions were 2.74, 3.00, and 3.41 log₁₀ for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.     

Pathogenesis of Chimeric MHV4/MHV-A59 Recombinant Viruses: the Murine Coronavirus Spike Protein Is a Major Determinant of Neurovirulence

The mouse hepatitis virus (MHV) spike glycoprotein, S, has been implicated as a major determinant of viral pathogenesis. In the absence of a full-length molecular clone, however, it has been difficult to address the role of individual viral genes in pathogenesis. By using targeted RNA recombination to introduce the S gene of MHV4, a highly neurovirulent strain, into the genome of MHV-A59, a mildly neurovirulent strain, we have been able to directly address the role of the S gene in neurovirulence. In cell culture, the recombinants containing the MHV4 S gene, S4R22 and S4R21, exhibited a small-plaque phenotype and replicated to low levels, similar to wild-type MHV4. Intracranial inoculation of C57BL/6 mice with S4R22 and S4R21 revealed a marked alteration in pathogenesis. Relative to wild-type control recombinant viruses (wtR13 and wtR9), containing the MHV-A59 S gene, the MHV4 S gene recombinants exhibited a dramatic increase in virulence and an increase in both viral antigen staining and inflammation in the central nervous system. There was not, however, an increase in the level of viral replication in the brain. These studies demonstrate that the MHV4 S gene alone is sufficient to confer a highly neurovirulent phenotype to a recombinant virus deriving the remainder of its genome from a mildly neurovirulent virus, MHV-A59. This definitively confirms previous findings, suggesting that the spike is a major determinant of pathogenesis.     

Origin and Possible Mode of Action of a Tissue Antagonist of Interferon

WE have presented data¹ supporting the hypothesis proposed by Taylor² and by Friedman and Sonnabend³ that interferon acts through a protein which confers the antiviral state on the cell. We postulated two separate regulatory mechanisms: one governing interferon synthesis and the other the production and action of the antiviral protein in the cell⁴. Our studies on the regulation of the antiviral state led us to the demonstration of a substance in human amniotic and chorionic membranes which, when added to the cell 4–18 h after interferon, decreased the antiviral state⁵. The extraction and purification of this tissue antagonist of interferon (TAI) have been previously described. TAI was resistant to proteases (trypsin, pepsin) and to nucleases (RNAase, DNAase). It was also resistant to heating at 56° C or 75° C for 1 h and was only inactivated at 95° C. Although the biologically active component was not chemically defined, its properties were reminiscent of mucopolysaccharides. It is unknown at present whether the TAI represents a substance or a group of substances.     

Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.The assembly of coronaviruses is driven principally by homotypic and heterotypic interactions between the two most abundant virion proteins, the membrane protein (M) and the nucleocapsid protein (N) (14, 32). The M protein is a triple-spanning transmembrane protein residing in the virion envelope, which is derived from the cellular budding site, the endoplasmic reticulum-Golgi intermediate compartment. More than half of the M molecule, its carboxy-terminal endodomain, is situated in the interior of the virion, where it contacts the nucleocapsid (46, 50). Also found in the virion envelope is the spike protein (S), which, although crucial for viral infectivity, is not an essential participant in assembly. The other canonical component of the coronavirus envelope is the small envelope protein (E), the function of which is enigmatic. Some evidence suggests that the E protein does not make sequence-specific contacts with other viral proteins (27) but instead functions by modifying the budding compartment, perhaps as an ion channel (56, 57). Alternatively, or additionally, E could act in a chaperone-like fashion to facilitate homotypic interactions between M protein monomers or oligomers (4).The N protein is the only protein constituent of the helically symmetric nucleocapsid, which is located in the interior of the virion. Coronavirus N proteins are largely basic phosphoproteins that share a moderate degree of homology across all three of the phylogenetic groups within the family (29). Some time ago, we proposed a model that pictured the N protein as comprising three domains separated by two spacers (A and B) (40). This arrangement was originally inferred from a sequence comparison of the N genes of multiple strains of the prototypical group 2 coronavirus, mouse hepatitis virus (MHV), and its validity seemed to be reinforced by numerous sequences that later became available. Part of this model, the delineation of spacer B and the acidic, carboxy-terminal domain 3, has been well supported by subsequent work (22, 25, 41, 42). However, a wealth of recent, detailed structural studies of bacterially expressed domains of the N proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) and of infectious bronchitis virus (IBV) has much more precisely mapped boundaries within the remainder of the N molecule (8, 16, 21, 23, 47, 51, 60). The latter studies have shown that the N protein contains two independently folding domains, designated the N-terminal domain (NTD) and the C-terminal domain (CTD). It should be pointed out that this nomenclature can be misleading: the NTD does not contain the amino terminus of the protein, and the CTD does not contain the carboxy terminus of the protein. Specifically, the CTD does not include spacer B and domain 3. The NTD and the CTD are separated by an intervening serine- and arginine-rich region; this region was previously noted to resemble the SR domains of splicing factors (42), and it has recently been shown to be intrinsically disordered (6, 7).In the assembled virion, the three known partners of the N protein are the M protein, the genomic RNA, and other copies of the N protein itself. We have sought to develop genetic and molecular biological methods that will begin to elucidate the varied ways in which the N molecule interacts during MHV infection. We previously found that the fusion of N protein domain 3 to a heterologous marker, green fluorescent protein (GFP), results in incorporation of GFP into virions (22). In the present study, we similarly fused each of the individual domains of N to GFP, and we thereby uncovered two strong modes of N protein-N protein interaction that likely contribute to virion architecture.     

The Spike Protein of Murine Coronavirus Mouse Hepatitis Virus Strain A59 Is Not Cleaved in Primary Glial Cells and Primary Hepatocytes

Mouse hepatitis virus strain A59 (MHV-A59) produces meningoencephalitis and severe hepatitis during acute infection. Infection of primary cells derived from the central nervous system (CNS) and liver was examined to analyze the interaction of virus with individual cell types derived from the two principal sites of viral replication in vivo. In glial cell cultures derived from C57BL/6 mice, MHV-A59 produces a productive but nonlytic infection, with no evidence of cell-to-cell fusion. In contrast, in continuously cultured cells, this virus produces a lytic infection with extensive formation of syncytia. The observation of few and delayed syncytia following MHV-A59 infection of hepatocytes more closely resembles infection of glial cells than that of continuously cultured cell lines. For MHV-A59, lack of syncytium formation correlates with lack of cleavage of the fusion glycoprotein, or spike (S) protein. The absence of cell-to-cell fusion following infection of both primary cell types prompted us to examine the cleavage of the spike protein. Cleavage of S protein was below the level of detection by Western blot analysis in MHV-A59-infected hepatocytes and glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. Thus, cleavage of the spike protein does not seem to be essential for entry and spread of the virus in vivo, as well as for replication in vitro.     

Localization of Action of the Is50-Encoded Transposase Protein

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp+), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp- IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules.