The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization. We observed that the psi (Ψ) packaging signal and the dimerization initiation sequence (DIS) had stabilizing effects on RSV Gag condensates, while RNAs lacking these features promoted or antagonized condensation, depending on local protein concentration and condensate architecture. An RNA containing Ψ, DIS, and the dimerization linkage structure (DLS) that is capable of stable dimer formation was observed to act as a bridge between RSV Gag condensates. These observations suggest additional, condensate-related roles for Gag-Ψ binding, gRNA dimerization, and Gag dimerization/multimerization in gRNA selection and packaging, representing a significant step forward in our understanding of how these interactions collectively facilitate efficient genome packaging.

Human coronavirus 229E (HCoV-229E) is an alpha coronavirus that infects humans and bats. In common with all positive-strand RNA viruses, 229E infection causes rearrangements of the host’s intracellular membranes to form replication organelles, a highly conserved and vital step in the viral replication cycle. Here, we investigated the role of the ESCRT protein VPS4A in 229E infection. We found that functional VPS4A was required for the formation of replication organelles and localizing viral RNA to these structures in host cells to facilitate viral genome replication. We validated this effect using small molecule inhibitors to VPS4A, significantly reducing virus replication. We also showed that other ESCRTS, like CHMP4B, were required for the virus replication step, whereas VPS37A was involved in the post-replication stages. The absence of a functional VPS4A prevented the remodeling of membranes to form viral replication centers and, therefore, exposed the viral RNA, triggering an inflammatory immune response as indicated by elevated levels of IL-6. Interestingly, we observed the role of VPS4A to be similar for the OC43 coronavirus, indicating it could be conserved across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of coronaviruses is imperative to finding more effective ways to control them.