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The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing viral replication to occur unhindered. Mutation or deletion of E3L severely affects viral host range and pathogenesis. While the monkeypox virus (MPXV) genome encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein with a truncation of 37 N-terminal amino acids. VACV with a genome encoding a similarly truncated E3L protein (VACV-E3LΔ37N) has been shown to be attenuated in mouse models, and infection with VACV-E3LΔ37N has been shown to lead to activation of the host antiviral protein kinase R pathway. In this report, we present data demonstrating that, despite containing a truncated E3 homologue, MPXV phenotypically resembles a wild-type (wt) VACV rather than VACV-E3LΔ37N. Thus, MPXV appears to contain a gene or genes that can suppress the phenotypes associated with an N-terminal truncation in E3. The suppression maps to sequences outside F3L, suggesting that the suppression is extragenic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue. IMPORTANCE Poxviruses have evolved to have many mechanisms to evade host antiviral innate immunity; these mechanisms may allow these viruses to cause disease. Within the family of poxviruses, variola virus (which causes smallpox) is the most pathogenic, while monkeypox virus is intermediate in pathogenicity between vaccinia virus and variola virus. Understanding the mechanisms of monkeypox virus innate immune evasion will help us to understand the evolution of poxvirus innate immune evasion capabilities, providing a better understanding of how poxviruses cause disease.

Monkeypox virus (MPXV) infection fails to activate the host anti-viral protein, PKR, despite lacking a full-length homologue of the vaccinia virus (VACV) PKR inhibitor, E3. Since PKR can be activated by dsRNA produced during a viral infection, we have analyzed the accumulation of dsRNA in MPXV-infected cells. MPXV infection led to less accumulation of dsRNA than VACV infection. Because in VACV infections accumulation of abnormally low amounts of dsRNA is associated with mutations that lead to resistance to the anti-poxvirus drug isatin beta-thiosemicarbazone (IBT), we investigated the effects of treatment of MPXV-infected cells with IBT. MPXV infection was eight-fold more resistant to IBT than wild-type vaccinia virus (wtVACV). These results demonstrate that MPXV infection leads to the accumulation of less dsRNA than wtVACV, which in turn likely leads to a decreased capacity for activation of the dsRNA-dependent host enzyme, PKR.

In the nineteenth century, smallpox ravaged through the United States and Canada. At this time, a botanical preparation, derived from the carnivorous plant Sarracenia purpurea, was proclaimed as being a successful therapy for smallpox infections. The work described characterizes the antipoxvirus activity associated with this botanical extract against vaccinia virus, monkeypox virus and variola virus, the causative agent of smallpox. Our work demonstrates the in vitro characterization of Sarracenia purpurea as the first effective inhibitor of poxvirus replication at the level of early viral transcription. With the renewed threat of poxvirus-related infections, our results indicate Sarracenia purpurea may act as another defensive measure against Orthopoxvirus infections.

Coronaviruses are enveloped viruses that assemble at intracellular membranes of the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) in infected cells.1 S and M are the main components of the viral envelope. The E protein is a minor component of the envelope, but plays an important role in virus assembly.2 Coronavirus envelope formation is nucleocapsid independent. Expression of only the E and M proteins is sufficient for the formation of virus-like-particles (VLPs) of many coronaviruses.3–5 The N protein is a multifunctional phosphoprotein that encapsidates the viral genome and plays a role in virus assembly.6, 7 N also appears to be involved in viral RNA replication and/or transcription.8, 9 The aim in this study was to provide a comprehensive view of the subcellular localization of the main SARS-CoV structural proteins. The S, M, E, and N genes were expressed in BHK-21 cells, and localization of the proteins was analyzed by indirect immunofluorescence microscopy. The proteins were co-analyzed with specific organelle markers for the endoplasmic reticulum (ER) and Golgi. Additionally, the M and E proteins were co-localized with a cellular marker for the ERGIC. The N protein remained cytoplasmic. The S, M, and E proteins were found to concentrate to the Golgi region, although some S appeared to also be transported to the cell surface.

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

To decrease virulence, we deleted the E3L gene, which is required for interferon-resistance and virulence, and replaced it with a gene from Ambystoma tigrinum virus (ATV, the new virus is NYVAC-C+12-ATV), which restores a single round of replication. Results In vitro characterization of the constructs demonstrates restoration of replication in primary and human cell lines. NYVAC-C+12-ATV leads to induction of pro-inflammatory signal transduction pathways. Pathogenicity studies in newborn mice demonstrate attenuation of 3–5 logs when compared to wt vaccinia virus or to NYCBH.