We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot‐and‐mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta‐glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT‐2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N‐terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.

Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6n5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell. Previous work has highlighted a histidine-α-helix charge-dipole interaction at the twofold axes of symmetry between pentamers and has suggested that this interaction plays a role in acid-induced disassembly. The validity of this theory has now been tested by converting the implicated residue, His-142 of protein 1C, to Arg, Phe and Asp. The effects of such changes were studied by using a previously described vaccinia virus expression system, in which synthesis and processing of FMDV capsid proteins results in the self-assembly of capsids. In agreement with the histidine-α-helix charge-dipole theory, assembly in the arginine mutant was found to be greatly reduced, while capsids of the aspartic acid mutant were considerably more stable under acidic conditions than the wild-type. Aberrant but acid-stable complexes were obtained in the phenylalanine mutant.

We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.VP22, the product of the UL49 gene of herpes simplex virus (8), is a major structural component of the virion. The protein is 301 residues in length, basic, and subject to a number of posttranslational modifications including phosphorylation (7) and nucleotidylylation (2). We previously reported that VP22 exhibits the unusual property of transport between cells (5). Transport was observed after introduction of the VP22 gene by several routes, including transfection or microinjection of the isolated gene in plasmid constructs or by infection with a nonreplicating herpesvirus encoding the native VP22 gene. One of the features of transport was that in cells actively synthesizing the protein, VP22 was located predominantly in the cytoplasm, where it could be observed in filamentous arrays colocalizing with bundled microtubules (4), while in the surrounding cells, VP22 was observed mainly in the nucleus, where it could also be observed colocalizing with chromatin in mitotic cells. A short C-terminal deletion mutant of VP22 lacking 34 residues was expressed normally and exhibited unaltered cytoplasmic localization in the primary cells expressing VP22 but failed to spread to the surrounding cells. Spread of VP22 was also sensitive to treatment of cells with cytochalasin D (5). In addition, we found that this transport activity was retained in a fusion protein consisting of VP22 linked to green fluorescent protein (GFP) which behaved essentially like the native protein with respect to expression, localization, and spread (5).We subsequently reported that trafficking of the GFP-VP22 fusion protein could not be readily observed in living cells (6), in agreement with the results of Fang and colleagues (9), but was detected in methanol-fixed cells either by examining intrinsic GFP fluorescence or by immunofluorescence (IF) analysis with anti-GFP antibodies (6). More recently, other laboratories have observed spread of a VP22-GFP fusion protein in fixed but ...

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

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