Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.nucleoprotein | nucleocapsid ring | atomic structure | paramyxovirus | ribonucleoprotein T he Paramyxoviridae family of nonsegmented negative-strand RNA viruses includes many serious pathogens of humans and animals including mumps virus, measles virus, parainfluenza viruses 1-5, Nipah virus, Hendra virus, and Newcastle disease virus, all of which are in the Paramyxovirinae subfamily, and respiratory syncytial virus (RSV) and human metapneumovirus, which are in the subfamily Pneumoviridae. The Paramyxoviridae belong in the order Mononegavirales of membrane enveloped negative-strand RNA viruses (NSV), an order that includes the Rhabdoviridae (rabies virus and vesicular stomatitis virus; VSV), Orthomyxoviridae (influenza virus) and Filoviridae (Ebola virus) (1). Parainfluenza virus 5 (PIV5) is a paramyxovirus that was initially isolated from rhesus monkey-kidney cell cultures (2) and although PIV5 is not known to cause human disease (3), it is used as a prototype in the study of paramyxoviruses.PIV5 has a nonsegmented single-stranded RNA genome of negative polarity of 15,246 nucleotides that encodes eight proteins (1): Three integral membrane proteins, fusion protein F and attachment protein hemagglutinin-neuraminidase (HN) and a small hydrophobic protein (SH). Inside the virion envelope lies a helical nucleocapsid core containing the R...

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8 + T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4 + T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

c Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.