Avian sarcoma/leukosis virus (RCAS)-mediated over-expression of IFITM3 protects chicks from highly pathogenic avian influenza virus subtype H5N1.

Broad-spectrum antiviral activities of interferon-induced transmembrane proteins (IFITMs) are primarily attributed to in vitro inhibition of viral entry. Here, we used an avian sarcoma-leukosis virus (RCAS)-based gene transfer system and successfully generated chicks that constitutively express chicken IFITM3 (chIFITM3). The chIFITM3-overexpressing chicks showed significant protection and disease tolerance against highly pathogenic avian influenza virus (HPAIV) H5N1 (Clade 2.2.1.2). The chicks, overexpressing chIFITM3, also showed delayed onset of clinical symptoms, reduced viral shedding, and alleviated histopathologic alterations compared to control and challenged chicks. These findings highlight that overexpression of chIFITM3 provide a substantial defense against zoonotic H5N1 in vivo.

Plectin promotes tumor formation by B16 mouse melanoma cells via regulation of Rous sarcoma oncogene activity.

BACKGROUND: Melanoma is a malignant tumor characterized by high proliferation and aggressive metastasis. To address the molecular mechanisms of the proto-oncogene, Rous sarcoma oncogene (Src), which is highly activated and promotes cell proliferation, migration, adhesion, and metastasis in melanoma. Plectin, a cytoskeletal protein, has recently been identified as a Src-binding protein that regulates Src activity in osteoclasts. Plectin is a candidate biomarker of certain tumors because of its high expression and the target of anti-tumor reagents such as ruthenium pyridinecarbothioamide. The molecular mechanisms by which plectin affects melanoma is still unclear. In this study, we examined the role of plectin in melanoma tumor formation. METHODS: We used CRISPR/Cas9 gene editing to knock-out plectin in B16 mouse melanoma cells. Protein levels of plectin and Src activity were examined by western blotting analysis. In vivo tumor formation was assessed by subcutaneous injection of B16 cells into nude mice and histological analysis performed after 2 weeks by Hematoxylin-Eosin (H&E) staining. Cell proliferation was evaluated by direct cell count, cell counting kit-8 assays, cyclin D1 mRNA expression and Ki-67 immunostaining. Cell aggregation and adhesion were examined by spheroid formation, dispase-based dissociation assay and cell adhesion assays. RESULTS: In in vivo tumor formation assays, depletion of plectin resulted in low-density tumors with large intercellular spaces. In vitro experiments revealed that plectin-deficient B16 cells exhibit reduced cell proliferation and reduced cell-to-cell adhesion. Since Src activity is reduced in plectin-deficient melanomas, we examined the relationship between plectin and Src signaling. Src overexpression in plectin knockout B16 cells rescued cell proliferation and improved cell-to-cell adhesion and cell to extracellular matrix adhesion. CONCLUSION: These results suggest that plectin plays critical roles in tumor formation by promoting cell proliferation and cell-to-cell adhesion through Src signaling activity in melanoma cells.

The expression profiles of chemokines, innate immune and apoptotic genes in tumors caused by Rous Sarcoma Virus (RSV-A) in chickens.

Innate immune genes play an important role in the immune responses to Rous sarcoma virus (RSV)-induced tumor formation and metastasis. Here, we determined in vivo expression of chemokines, innate immune and apoptotic genes in Synthetic Broiler Dam Line (SDL) chickens following RSV-A infection. The mRNA expression of genes was determined at the primary site of infection and in different organs of progressor, regressor and non-responder chicks, using RT-qPCR. Our results indicated a significant upregulation of: (1) chemokines, such as MIP1ß and RANTES, (2) the innate immune gene TLR4, and (3) p53, a tumor-suppressor gene, at the site of primary infection in progressor chickens. In contrast, inducible nitric oxide synthase (iNOS) gene expression was significantly downregulated in progressor chicks compared to uninfected, control chicks. All of the innate immune genes were significantly upregulated in the lungs and liver of the progressor and regressor chicks compared to control chicks. In the spleen of progressor chicks, RANTES, iNOS and p53 gene expression were significantly increased, whereas MIP1ß and TLR4 gene expression was significantly downregulated, compared to control chicks. The lungs and livers of non-responder chicks expressed a low level of iNOS and MIP1ß, whereas RANTES, TLR4, and p53 gene expression were significantly upregulated compared to uninfected control chicks. In addition, there was a significant downregulation of RANTES, MIP1ß, and TLR4 gene expression in non-responder chicks. These results suggest the different response to infection of chicks with RSV-A is due to differential changes in the expression of innate immune genes in different organs.

Identification of novel microRNAs in Rous sarcoma Virus (RSV) and their target sites in tumor suppressor genes of chicken.

A small non-coding, evolutionarily conserved regulatory RNA molecule known as microRNA (miRNA) regulates various cellular activities and pathways. MicroRNAs remain evolutionarily conserved in different species of same taxa. They are present in all organisms including viruses. Viral miRNAs are small, less conserved and less stable and have higher negative minimal folding free energy than miRNAs of different organisms. The size of viral precursor miRNA is approximately 60-119 nucleotides in length. The structure of the mature miRNA sequences is predicted by using higher negative MFE (ΔG) value. Rous sarcoma Virus (RSV), named after its inventor Peyton Rous, has been known for causing tumors in the chicken for which it is known as an oncogenic retrovirus. Using specific criteria we have predicted 5 potential miRNAs in RSV which targeted 8 tumor suppressor genes in Gallus gallus. This study aims to predict the potential miRNAs, secondary structures and their targets for better understanding of the regulatory network of Rous sarcoma virus miRNA in forming sarcoma.

Research Note: Rous sarcoma growth differs among congenic lines containing major histocompatibility (B) complex recombinants.

New arrangements of chicken major histocompatibility complex (MHC) class I BF and class IV BG genes are created through recombination. Characterizing the immune responses of such recombinants reveals genes or gene regions that contribute to immunity. Inbred Line UCD 003 (B17B17) served as the genetic background for congenic lines, each containing a unique MHC recombinant. After an initial cross to introduce a specific recombinant, 10 backcrosses to the inbred line produced lines with 99.9% genetic uniformity. The current study compared Rous sarcoma virus (RSV) tumor growth in 5 congenic lines homozygous for MHC recombinants (003.R1 = BF24-BG23, 003.R2 = BF2-BG23, 003.R4 = BF2-BG23, 003.R5 = BF21-BG19, and 003.R13 = BF17-BG23). Two experiments used a total of 70 birds from the 5 congenic lines inoculated with 20 pock forming units of RSV subgroup C at 6 wk of age. Tumor size was scored 6 times over 10 wk postinoculation followed by assignment of a tumor profile index (TPI) based on the tumor size scores. Tumor growth over time and rank transformed TPI values were analyzed by least squares ANOVA. Tumor size increased over the experimental period in all genotypes through 4 wk postinoculation. After this time, tumor size increased in Lines 003.R1, plateaued in Lines 003.R2, 003.R4, and 003.R13, and declined in 003.R5. Tumor growth over time was significantly lower in Line 003.R5 compared with all other genotypes. In addition, Line 003.R5 chickens had significantly lower TPI values compared with Lines 003.R2, 003.R4, and 003.R13. The TPI of Line 003.R1 did not differ significantly from any of the other genotypes. The BF21 in Line 003.R5 produced a greater response against subgroup C RSV tumors than did BF24, found in 003.R1; BF2 found in 003.R2 and R4 as well as BF17 found in 003.R13.

The road from Rous sarcoma virus to precision medicine.

In 1911, more than a century ago, Peyton Rous described a curious observation, later explained by a virus named for him that led to the discovery of oncogenes, the modern era of cancer research, and the emergent field of precision medicine (1911. J. Exp. Med. https://doi.org/10.1084/jem.13.4.397).

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a "Ψ" packaging signal located in the gRNA 5'-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5'-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag-RNA interactions.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Reverse Engineering Provides Insights on the Evolution of Subgroups A to E Avian Sarcoma and Leukosis Virus Receptor Specificity.

The initial step of retrovirus entry-the interaction between the virus envelope glycoprotein trimer and a cellular receptor-is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as receptors. The highly homologous subgroup A to E Avian Sarcoma and Leukosis Virus (ASLV) glycoproteins belong to the group of class 1 viral fusion proteins with a two-step triggering mechanism that allows experimental access to intermediate structures during the fusion process. We and others have taken advantage of replication-competent ASLVs and exploited genetic selection strategies to force the ASLVs to naturally evolve and acquire envelope glycoprotein mutations to escape the pressure on virus entry and still yield a functional replicating virus. This approach allows for the simultaneous selection of multiple mutations in multiple functional domains of the envelope glycoprotein that may be required to yield a functional virus. Here, we review the ASLV family and experimental system and the reverse engineering approaches used to understand the evolution of ASLV receptor usage.

Avian Sarcoma and Leukosis Virus Envelope Glycoproteins Evolve to Broaden Receptor Usage Under Pressure from Entry Competitors †.

The subgroup A through E avian sarcoma and leukosis viruses (ASLV(A) through ASLV(E)) are a group of highly related alpharetroviruses that have evolved their envelope glycoproteins to use different receptors to enable efficient virus entry due to host resistance and/or to expand host range. Previously, we demonstrated that ASLV(A) in the presence of a competitor to the subgroup A Tva receptor, SUA-rIgG immunoadhesin, evolved to use other receptor options. The selected mutant virus, RCASBP(A)Δ155-160, modestly expanded its use of the Tvb and Tvc receptors and possibly other cell surface proteins while maintaining the binding affinity to Tva. In this study, we further evolved the Δ155-160 virus with the genetic selection pressure of a soluble form of the Tva receptor that should force the loss of Tva binding affinity in the presence of the Δ155-160 mutation. Viable ASLVs were selected that acquired additional mutations in the Δ155-160 Env hypervariable regions that significantly broadened receptor usage to include Tvb and Tvc as well as retaining the use of Tva as a receptor determined by receptor interference assays. A similar deletion in the hr1 hypervariable region of the subgroup C ASLV glycoproteins evolved to broaden receptor usage when selected on Tvc-negative cells.

Cytokine gene expression following RSV-A infection.

The present study determines the cytokine gene expression in chickens following RSV-A infection, using RT-qPCR. In susceptible chickens tumors progressed to  fulminating metastatic tumors while it regressed in  regressors  chickens and some resistant non-responder chickens did not respond to RSV-A infection and thus did not develop tumors at all. The in vivo expression of pro-inflammatory cytokines, Th1 cytokines and Th2 cytokines was determined at the primary site of infection, as well as in different organs of progressor, regressor and non-responder chicks at different time intervals. Our results indicated a significant upregulation of the pro-inflammatory cytokines, IL-6 and IL-8, in all the organs of progressor chicks, while they were significantly lower in regressor and non-responder chicks. The expression of the Th1 cytokines IFN-γ and TNF-α was low in all of the organs of the progressor group, except that in  spleen. In contrast, regressor and non-responder groups showed high expression of IFN-γ and TNF-α. Further, there was an early upregulation of the expression of the Th2 cytokine, IL-10, TGF-ß and GM-CSF, in all of the organs of progressors as compared to uninfected control.

MHC-B haplotypes impact susceptibility and resistance to RSV-A infection.

We investigated the impact of haplotype of major histocompatibility complex (MHC)-B on the outcome of infection of Synthetic Dam Line (SDL) broiler strain with Rous Sarcoma Virus (RSV). Genomic analysis of MHC-B haplotypes, revealed a total of 12 known standard haplotypes that constituted to twenty-five different genotypes and one new haplotype of 217 bp size, designated BX. The inoculation of RSV-A in SDL chicks resulted in the development of tumors of progressive or regressive phenotypes with varying tumor profile index (TPI). Haplotypes B2, B21 and B22had low TPI scores (1 or 2) with less mortality and were resistant to RSV-A tumor. The haplotypes B13, B13.1., B15, B15.1. and B15.2. had significantly higher TPI scores (5 or 6), indicating a susceptibility to RSV-A. The genotype, Bx /Bx, had a mean TPI score of 3.67 ± 1.33, which was closer to the resistant haplotype. Sequence analysis of the new haplotype (BX) revealed 99.5% similarity with B2 haplotype. Metastases was observed in 44% of chicks and comprised of mixed fibrosarcoma and myxosarcoma.

Remembering Jan Svoboda: A Personal Reflection.

The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term persistence of the viral genome in non-productive mammalian cells, and he supported the DNA provirus hypothesis of Howard Temin. He showed how the virus can be rescued in the infectious form and elucidated the replication-competent nature of the Prague strain of RSV later used for the identification of the src oncogene. His studies straddled molecular oncology and virology, and he remained an active contributor to the field until his death last year. Throughout the 50 years that I was privileged to know Svoboda as my mentor and friend, I admired his depth of scientific inquiry and his steadfast integrity in the face of political oppression.

Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines.

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

A C-terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly.

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

Cell Association in Rous Sarcoma Virus (RSV) Rescue and Cell Infection.

In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.

Molecular events accompanying rous sarcoma virus rescue from rodent cells and the role of viral gene complementation.

UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.