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Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here, we demonstrate that OPCs, but not OLs, are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival, proliferation, or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that, while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains, where FrCasEinfected NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. [ABSTRACT FROM AUTHOR]

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi’s sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi’s sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers. [ABSTRACT FROM AUTHOR]

Developing broad-spectrum vaccines and optimal vaccination strategies is crucial to controlling the COVID-19 pandemic. Here, we generated a chimpanzee adenoviral vector-based COVID-19 vaccine carrying broad-spectrum immunogens, modified full-length spike, and conserved T-cell epitopes of SARS-CoV-2, and assessed its immune response in mice through intramuscular (i.m.), intranasal (i.n.), or combined immunization routes (i.m. + i.n., or i.n. + i.m.). Compared to other vaccination strategies, the two combined regimens elicited higher neutralizing antibody (NAb) responses to all variants. Compared to i.n. + i.m. regimen, the i.m. + i.n. regimen stimulated a stronger secondary GC response, which is more pivotal to high-quality antibody production than the primary GC response. Moreover, the i.m. + i.n. regimen was adept at mediating systemic cellular immunity, while the i.n. + i .m. regimen tended to elicit lung tissue-resident memory T (TRM) cell responses. Overall, the two combined regimens induced comprehensive but distinct immune responses consisting of lgA, lgG, NAbs, GC B cells, long-lived plasma cells, TRM cells, and systemic memory T cells, which conferred complete protection against BA.2 infection in hACE2 transgenic mice, and warranted further investigation as potential universal vaccination strategies. IMPORTANCE The development of broad-spectrum SARS-CoV-2 vaccines will reduce the global economic and public health stress from the COVID-19 pandemic. The use of conserved T-cell epitopes in combination with spike antigen that induce humoral and cellular immune responses simultaneously may be a promising strategy to further enhance the broad spectrum of COVID-19 vaccine candidates. Moreover, this research suggests that the combined vaccination strategies have the ability to induce both effective systemic and mucosal immunity, which may represent promising strategies for maximizing the protective efficacy of respiratory virus vaccines. [ABSTRACT FROM AUTHOR]

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans-complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the "non-neurovirulent" amphotropic helper virus, 4070A, or pgag-polgpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration. [ABSTRACT FROM AUTHOR]

Aggressive B-cell lymphoma is one of the most common types of blood malignancy. Robust delivery of genes of interest into target cells, long-term gene expression, and minimal risk of secondary effects are highly desirable for translational medicine including gene therapy and studies on gene function. However, efficient gene delivery into viral or nonviral B-lymphoma cells remains a challenge. Here, we report a strategy for inducing foreign gene expression in B-lymphoma cells by using a vector based on the novel parainfluenza virus PIV5-L (a strain isolated from B cells) that enabled us to study and control the function of a gene product within B-lymphoma cells. Using enhanced green fluorescent protein (eGFP) as a reporter, we successfully rescued PIV5-L and established a one-step system to generate PIV5-L virus-like particles (L-VLPs) with efficient delivery into a broad spectrum of susceptible B-lymphoma cell lines, including Epstein-Barr virus (EBV)- or Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed B-lymphoblastoid cells. Similar to lentiviral vector, the L-VLP highly expressed exogenous genes and remained stable for long periods without obvious negative effects on cell viability. Taken together, these data demonstrate that the PIV5-L-based system provides a potential new strategy for the delivery of desirable genes and the treatment of cancer. [ABSTRACT FROM AUTHOR]

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3 of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed, by using a Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008, 3060, and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1-3 as well as between the internal VP4 and the three external capsid proteins of FMDV might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance, as compared to the Y2079-encoding viruses. Interestingly, none of all the 16 recued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s), and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV. [ABSTRACT FROM AUTHOR]

All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes. [ABSTRACT FROM AUTHOR]

Chemokines and their receptors play important roles in normal physiological functions and the pathogeneses of a wide range of human diseases, including the entry of human immunodeficiency virus type 1 (HIV-1). However, the use of natural chemokines to probe receptor biology or to develop therapeutic drugs is limited by their lack of selectivity and the poor understanding of mechanisms in ligand-receptor recognition. We addressed these issues by combining chemical and structural biology in research into molecular recognition and inhibitor design. Specifically, the concepts of chemical biology were used to develop synthetically and modularly modified (SMM) chemokines that are unnatural and yet have properties improved over those of natural chemokines in terms of receptor selectivity, affinity, and the ability to explore receptor functions. This was followed by using structural biology to determine the structural basis for synthetically perturbed ligand-receptor selectivity. As a proof-of-principle for this combined chemical and structural-biology approach, we report a novel D-amino acid-containing SMM-chemokine designed based on the natural chemokine called viral macrophage inflammatory protein II (vMIP-II). The incorporation of unnatural D-amino acids enhanced the affinity of this molecule for CXCR4 but significantly diminished that for CCR5 or CCR2, thus yielding much more selective recognition of CXCR4 than wild-type vMIP-II. This D-amino acid-containing chemokine also showed more potent and specific inhibitory activity against HIV-1 entry via CXCR4 than natural chemokines. Furthermore, the high-resolution crystal structure of this D-amino acid-containing chemokine and a molecular-modeling study of its complex with CXCR4 provided the structure-based mechanism for the selective interaction between the ligand and chemokine receptors and the potent anti-HIV activity of D-amino acid-containing chemokines. [ABSTRACT FROM AUTHOR]