Reduced vacuolar ATPase protects mice from Friend virus infection - an unintended but instructive effect in Hif-2afl mice.

During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.

FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Effects of antibiotic treatment on microbiota, viral transmission and viral pathogenesis of MoMuLV ts1 infected BALB/c mice.

The effects of normal and altered intestinal microbiota on murine retroviral transmission via the gastrointestinal tract (GIT) are diverse. The role of orally administered antibiotic treatment (ABX) on viral transmission, GIT microbial dysbiosis and subsequent pathogenesis of Moloney Murine Leukemia virus-temperature sensitive 1 (ts1) on BALB/c mice were studied. BALB/c mice were divided into four groups: ABXts1-Treatment/Infection;ABX-Treatment/No infection;ts1-No treatment/Infection;Ctrl (control)-No treatment/No infection. ABXts1 and ABX groups showed a significant phylogenetic shift (ANOSIM p-value = 0.001) in alpha and beta diversity comparisons for microbial community composition compared to Ctrl group. Mice in the ABXts1 and ABX groups showed megacolon compared to ts1 and Ctrl groups; ABXts1 and ts1 groups showed hepatosplenomegaly, thymus enlargement, and mesenteric lymphadenopathy compared to ABX and Ctrl groups. Ctrl group had no abnormal manifestations. ABX treatment and ts1 infection uniquely affect microbial community when compared to control: ABXts1 and ABX groups significantly reduce microbiome diversity by over 80% and ts1 group by over 30%. ABXts1 and ts1 groups' viral load and clinical manifestations of infection were comparable; antibiotic treatment did not notably affect ts1 infection. Transmission and pathophysiology of ts1 infection were not significantly altered by the microbial composition of the GI tract, but ts1 viral infection did result in microbial dysbiosis independent of antibiotic treatment.

Dynamics of Polarized Macrophages and Activated CD8+ Cells in Heart Tissue of Atlantic Salmon Infected With Piscine Orthoreovirus-1.

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8+ and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8+ granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8+ cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection.

Novel Host Protein TBC1D16, a GTPase Activating Protein of Rab5C, Inhibits Prototype Foamy Virus Replication.

Prototype foamy virus (PFV) is a member of the oldest family of retroviruses and maintains lifelong latent infection in the host. The lifelong latent infection of PFV may be maintained by the restriction factors of viral replication in the host. However, the mechanisms involved in PFV latent infection are poorly understood. Here, we found that TBC1D16, a TBC domain-containing protein, is significantly down-regulated after PFV infection. Tre2/Bub2/Cdc16 (TBC) domain-containing proteins function as Rab GTPase-activating proteins (GAPs) and are participates in the progression of some diseases and many signaling pathways. However, whether TBC proteins are involved in PFV replication has not been determined. Here, we found that TBC1D16 is a novel antiviral protein that targets Rab5C to suppress PFV replication. Overexpression TBC1D16 inhibited the transcription and expression of Tas and Gag, and silencing TBC1D16 enhanced the PFV replication. Moreover, the highly conserved amino acid residues R494 and Q531 in the TBC domain of TBC1D16 were essential for inhibiting PFV replication. We also found that TBC1D16 promoted the production of PFV-induced IFN-ß and the transcription of downstream genes. These results suggest that TBC1D16 might be the first identified TBC proteins that inhibited PFV replication and the mechanism by which TBC1D16 inhibited PFV replication could provide new insights for PFV latency.

In vivo imaging of retrovirus infection reveals a role for Siglec-1/CD169 in multiple routes of transmission.

Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer's patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection.

Loss of sphingosine kinase 1 increases lung metastases in the MMTV-PyMT mouse model of breast cancer.

Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.

The Interplay between ESCRT and Viral Factors in the Enveloped Virus Life Cycle.

Viruses are obligate parasites that rely on host cellular factors to replicate and spread. The endosomal sorting complexes required for transport (ESCRT) system, which is classically associated with sorting and downgrading surface proteins, is one of the host machineries hijacked by viruses across diverse families. Knowledge gained from research into ESCRT and viruses has, in turn, greatly advanced our understanding of many other cellular functions in which the ESCRT pathway is involved, e.g., cytokinesis. This review highlights the interplay between the ESCRT pathway and the viral factors of enveloped viruses with a special emphasis on retroviruses.

Bmp8a is an essential positive regulator of antiviral immunity in zebrafish.

Bone morphogenetic protein (BMP) is a kind of classical multi-functional growth factor that plays a vital role in the formation and maintenance of bone, cartilage, muscle, blood vessels, and the regulation of adipogenesis and thermogenesis. However, understanding of the role of BMPs in antiviral immunity is still limited. Here we demonstrate that Bmp8a is a newly-identified positive regulator for antiviral immune responses. The bmp8a-/- zebrafish, when infected with viruses, show reduced antiviral immunity and increased viral load and mortality. We also show for the first time that Bmp8a interacts with Alk6a, which promotes the phosphorylation of Tbk1 and Irf3 through p38 MAPK pathway, and induces the production of type I interferons (IFNs) in response to viral infection. Our study uncovers a previously unrecognized role of Bmp8a in regulation of antiviral immune responses and provides a target for controlling viral infection.

The Battle between Retroviruses and APOBEC3 Genes: Its Past and Present.

The APOBEC3 family of proteins in mammals consists of cellular cytosine deaminases and well-known restriction factors against retroviruses, including lentiviruses. APOBEC3 genes are highly amplified and diversified in mammals, suggesting that their evolution and diversification have been driven by conflicts with ancient viruses. At present, lentiviruses, including HIV, the causative agent of AIDS, are known to encode a viral protein called Vif to overcome the antiviral effects of the APOBEC3 proteins of their hosts. Recent studies have revealed that the acquisition of an anti-APOBEC3 ability by lentiviruses is a key step in achieving successful cross-species transmission. Here, we summarize the current knowledge of the interplay between mammalian APOBEC3 proteins and viral infections and introduce a scenario of the coevolution of mammalian APOBEC3 genes and viruses.

Changes in oxidation-antioxidation function on the thymus of chickens infected with reticuloendotheliosis virus.

BACKGROUND: Reticuloendotheliosis virus (REV) is a retrovirus that causes severe immunosuppression in poultry. Animals grow slowly under conditions of oxidative stress. In addition, long-term oxidative stress can impair immune function, as well as accelerate aging and death. This study aimed to elucidate the pathogenesis of REV from the perspective of changes in oxidative-antioxidative function following REV infection. METHODS: A total of 80 one-day-old specific pathogen free (SPF) chickens were randomly divided into a control group (Group C) and an REV-infected group (Group I). The chickens in Group I received intraperitoneal injections of REV with 104.62/0.1 mL TCID50. Thymus was collected on day 1, 3, 7, 14, 21, 28, 35, and 49 for histopathology and assessed the status of oxidative stress. RESULTS: In chickens infected with REV, the levels of H2O2 and MDA in the thymus increased, the levels of TAC, SOD, CAT, and GPx1 decreased, and there was a reduction in CAT and Gpx1 mRNA expression compared with the control group. The thymus index was also significantly reduced. Morphological analysis showed that REV infection caused an increase in the thymic reticular endothelial cells, inflammatory cell infiltration, mitochondrial swelling, and nuclear damage. CONCLUSIONS: These results indicate that an increase in oxidative stress enhanced lipid peroxidation, markedly decreased antioxidant function, caused thymus atrophy, and immunosuppression in REV-infected chickens.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

Differences and commonalities in plasma membrane recruitment of the two morphogenetically distinct retroviruses HIV-1 and MMTV.

Retroviral Gag polyproteins are targeted to the inner leaflet of the plasma membrane through their N-terminal matrix (MA) domain. Because retroviruses of different morphogenetic types assemble their immature particles in distinct regions of the host cell, the mechanism of MA-mediated plasma membrane targeting differs among distinct retroviral morphogenetic types. Here, we focused on possible mechanistic differences of the MA-mediated plasma membrane targeting of the B-type mouse mammary tumor virus (MMTV) and C-type HIV-1, which assemble in the cytoplasm and at the plasma membrane, respectively. Molecular dynamics simulations, together with surface mapping, indicated that, similarly to HIV-1, MMTV uses a myristic switch to anchor the MA to the membrane and electrostatically interacts with phosphatidylinositol 4,5-bisphosphate to stabilize MA orientation. We observed that the affinity of MMTV MA to the membrane is lower than that of HIV-1 MA, possibly related to their different topologies and the number of basic residues in the highly basic MA region. The latter probably reflects the requirement of C-type retroviruses for tighter membrane binding, essential for assembly, unlike for D/B-type retroviruses, which assemble in the cytoplasm. A comparison of the membrane topology of the HIV-1 MA, using the surface-mapping method and molecular dynamics simulations, revealed that the residues at the HIV-1 MA C terminus help stabilize protein-protein interactions within the HIV-1 MA lattice at the plasma membrane. In summary, HIV-1 and MMTV share common features such as membrane binding of the MA via hydrophobic interactions and exhibit several differences, including lower membrane affinity of MMTV MA.

Transmission of Koala Retrovirus from Parent Koalas to a Joey in a Japanese Zoo.

Koala retrovirus (KoRV) is of an interest to virologists due to its currently active endogenization into the koala (Phascolarctos cinereus) genome. Although KoRV has frequently been isolated in wild and captive koala populations, its pathogenesis and transmission remain to be fully characterized, and most previous research has concentrated on adult koalas rather than on joeys. Here, we characterized KoRV isolates obtained from a deceased male joey and its parents (animals reared in a Japanese zoo) to investigate KoRV transmission mode and pathogenesis. We sequenced the KoRV long terminal repeat (LTR) and envelope genes isolated from the joey and its parents and found KoRV-A and KoRV-C in genomic DNA from both the parents and the joey. Notably, both parents were also positive for KoRV-B, whereas the joey was KoRV-B negative, further confirming that KoRV-B is an exogenous strain. The KoRV LTR sequence of the joey was considerably closer to that of its sire than its dam. For further characterization, total KoRV, KoRV-A, KoRV-B, and KoRV-C proviral loads were quantified in peripheral blood mononuclear cells from the parents and in blood samples from the joey. Total KoRV, KoRV-A, and KoRV-C proviral loads were also quantified for different tissues (bone, liver, kidney, lung, spleen, heart, and muscle) from the joey, revealing differences suggestive of a distinct tissue tropism (highest total KoRV proviral load in the spleen and lowest in bone). The amount of KoRV-C in the parents was less than that in the joey. Our findings contribute to an improved understanding of KoRV pathogenesis and transmission mode and highlight useful areas for future research.IMPORTANCE KoRV is unique among retroviruses in that one strain (KoRV-A) is undergoing endogenization, whereas the other main subtype (KoRV-B) and another subtype (KoRV-C) are reportedly exogenous strains. Its transmission and pathogenesis are of interest in the study of retroviruses and are crucial for any conservation strategy geared toward koala health. This study provides new evidence on the modes of KoRV transmission from parent koalas to their joey. We found vertical transmission of KoRV-A, confirming its endogenization, but with closer conservation between the joey and its sire than its dam (previous reports on joeys are rare but have postulated dam-to-joey vertical transmission). This is also the first report of a KoRV-B-negative joey from KoRV-B-positive parents, contrasting with the few previous reports of 100% transmission of KoRV-B from dams to joeys. Thus, the results in this study give some novel insights for the transmission mode of KoRV.

IFITM3 Reduces Retroviral Envelope Abundance and Function and Is Counteracted by glycoGag.

Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3.IMPORTANCE The viral envelope glycoprotein, known as "Env" in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.

Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus.

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus-infected cells are well known to release soluble viral proteins, defective viruses, and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small-particle flow cytometry, is an emerging analytical method enabling high-throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here, we analyzed the basic properties of Moloney MLV (M-MLV). We show that <24% of the total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contains a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus while highlighting the benefits of flow virometry for the field of virology.IMPORTANCE Gammaretroviruses, or, more specifically, murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analyzed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90 nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here, we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.

Proteome analysis of reticuloendotheliosis-virus-infected chicken embryo fibroblast cells through iTRAQ-based quantitative proteomics.

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.