Robinson, B. A. | bioRxiv Science

bioRxiv2018

Caspase-mediated cleavage of Murine Norovirus NS1/2 potentiates apoptosis and is required for persistent infection

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after infection. Murine norovirus (MNV) is also shed persistently in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of NS1 in promoting persistence is regulated by apoptosis. Following induction of apoptosis in infected cells, a minority of NS1 is cleaved from the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. MNV strain CR6 with these mutations (CR6{triangleup}casp) is profoundly compromised in infection of IECs and persistence in the intestine. Conversely, replication in tissues outside of the intestine, or in a cultured macrophage cell line, is unchanged, indicating that the requirement of NS1/2 cleavage is intestine-specific. Intriguingly, we also find that cleavage of CR6 NS1/2 potentiates apoptosis, suggesting that regulation of cell death is a novel function of this viral protein. Together, these data indicate that the ability of NS1 to promote MNV persistence in IECs is regulated by host caspases, and suggest that potentiation of apoptosis plays a role in viral tropism in the intestine.\n\nAuthor SummaryHuman Norovirus infection is highly contagious and the most common cause of acute gastroenteritis. Norovirus can persist and be shed for months after infection, leading to continued outbreaks. There are many unanswered questions as to host and viral components of norovirus pathogenesis that can be addressed within the murine norovirus (MNV) model system. We previously identified a critical role for a viral protein, NS1, for intestinal persistence. Herein we describe how the regulation of NS1 is critical for persistent infection in intestinal epithelial cells, but is not required for acute infection of non-epithelial cells, or infection of tissues outside of the gut. Additionally, we demonstrate that NS1 is both regulated by the host cell death machinery, and also reciprocally regulates that machinery to promote cell death during MNV infection, and found that this is specific to persistent strain of MNV. Altogether these data identify a role for how NS1 in a new pathway involved in establishing a persistent norovirus infection in the intestine.

microbiology↗

bioRxiv2017

HIV-1 initiates genomic RNA packaging in a unique subset of host RNA granules

How HIV-1 genomic RNA (gRNA) is packaged into assembling virus remains unclear. Here, we use biochemical and in situ approaches to identify the complex in which the capsid protein Gag first associates with gRNA, termed the packaging initiation complex. First, we show that in the absence of assembling Gag, non-nuclear non-translating gRNA is nearly absent from the soluble fraction of provirus-expressing cells, and is found instead primarily in complexes >30S. When we express a Gag mutant known to be arrested at packaging initiation, we find only one complex containing Gag and gRNA; thus, this complex corresponds to the packaging initiation complex. This [~]80S complex also contains two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6, and therefore corresponds to a co-opted host RNA granule and a previously described capsid assembly intermediate. Additionally, we find this granule-derived packaging initiation complex in HIV-1-infected H9 T cells, and demonstrate that wild-type Gag forms both the packaging initiation complex and a larger granule-derived complex corresponding to a late packaging/assembly intermediate. We also demonstrate that packaging initiation complexes are far more numerous than P bodies in situ. Finally, we show that Gag enters the [~]80S granule to form the packaging initiation complex via a two-step mechanism. In a step that is independent of a gRNA-binding domain, Gag enters a broad class of RNA granules, most of which lack gRNA. In a second step that is dependent on the gRNA-binding nucleocapsid domain of Gag or a heterologous gRNA-binding domain, Gag enters a gRNA-containing subset of these granules. Thus, we conclude that packaging in cells does not result from random encounters between Gag and gRNA; instead our data support a fundamentally different model in which Gag is directed to gRNA within a unique host RNA granule to initiate this critical event in HIV-1 replication.\n\nNontechnical SummaryTo form infectious virus, the HIV-1 capsid protein Gag must associate with and package the viral genomic RNA (gRNA) during the virus assembly process. HIV-1 Gag first associates with gRNA in the cytoplasm, forming a complex termed the packaging initiation complex; this complex subsequently targets to the plasma membrane where Gag completes the assembly and packaging process before releasing the virus from the cell. Although the packaging initiation complex is critical for infectious virus formation, its identity and composition, and the mechanism by which it is formed, remain unknown. Here we identify the packaging initiation complex, and demonstrate that it corresponds to a host RNA granule that is co-opted by the virus. RNA granules are diverse complexes utilized by host cells for all aspects of RNA storage and metabolism besides translation. Our study also defines the mechanism by which HIV-1 Gag enters this host RNA granule to form the packaging initiation complex, and reveal that it involves two steps that depend on different regions of Gag. Our finding that Gag co-opts a poorly studied host complex to first associate with gRNA during packaging provides a new paradigm for understanding this critical event in the viral life cycle.

microbiology↗