The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided \'specificity\' conferred by the totality of TRIM5α\'s additional domains that may account for its recently identified promiscuity.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Sterile alpha motif domain-containing proteins 9 and 9-like (SAMD9/9L) are associated with life-threatening genetic diseases in humans and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated human SAMD9L restricts HIV-1 in the late phases of replication, at the posttranscriptional and prematuration steps, impacting viral translation and, possibly, endosomal trafficking. Surprisingly, the paralog SAMD9 exerted an opposite effect, enhancing HIV-1. More broadly, we showed that SAMD9L restricts primate lentiviruses, but not a gammaretrovirus (MLV), nor 2 RNA viruses (arenavirus MOPV and rhabdovirus VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a conserved Schlafen-like active site necessary for HIV-1 restriction by human and a rodent SAMD9L. By testing a gain-of-function constitutively active variant from patients with SAMD9L-associated autoinflammatory disease, we determined that SAMD9L pathogenic functions also depend on the Schlafen-like active site. Finally, we found that the constitutively active SAMD9L strongly inhibited HIV, MLV, and, to a lesser extent, MOPV. This suggests that the virus-specific effect of SAMD9L may involve its differential activation/sensing and the virus ability to evade from SAMD9L restriction. Overall, our study identifies SAMD9L as an HIV-1 antiviral factor from the cell autonomous immunity and deciphers host determinants underlying the translational repression. This provides novel links and therapeutic avenues against viral infections and genetic diseases.

BACKGROUND: Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood.
RESULTS: To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS.
CONCLUSIONS: These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

The TRIM family is associated with the membrane, and its involvement in the progression, growth, and development of various cancer types has been researched extensively. However, the role played by the TRIM5 gene within this family has yet to be explored to a great extent in terms of hepatocellular carcinoma (HCC). The data of patients relating to mRNA expression and the survival rate of individuals diagnosed with HCC were extracted from The Cancer Genome Atlas (TCGA) database. UALCAN was employed to examine the potential link between TRIM5 expression and clinicopathological characteristics. In addition, enrichment analysis of differentially expressed genes (DEGs) was conducted as a means of deciphering the function and mechanism of TRIM5 in HCC. The data in the TCGA and TIMER2.0 databases was utilized to explore the correlation between TRIM5 and immune infiltration in HCC. WGCNA was performed as a means of assessing TRIM5-related co-expressed genes. The \"OncoPredict\" R package was also used for investigating the association between TRIM5 and drug sensitivity. Finally, qRT-PCR, Western blotting (WB) and immunohistochemistry (IHC) were employed for exploring the differential expression of TRIM5 and its clinical relevance in HCC. According to the results that were obtained from the vitro experiments, mRNA and protein levels of TRIM5 demonstrated a significant upregulation in HCC tissues. It is notable that TRIM5 expression levels were found to have a strong association with the infiltration of diverse immune cells and displayed a positive correlation with several immune checkpoint inhibitors. The TRIM5 expression also displayed promising clinical prognostic value for HCC patients.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual\'s life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer\'s disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

The innate immune system of mammals is formed by a complex web of interacting proteins, which together constitute the first barrier of entry for infectious pathogens. Genes from the E3-ubiquitin ligase tripartite motif (TRIM) family have been shown to play an important role in the innate immune system by restricting the activity of different retrovirus species. For example, TRIM5 and TRIM22 have both been associated with HIV restriction and are regarded as crucial parts of the antiretroviral machinery of mammals. Our analyses of positive selection corroborate the great significance of these genes for some groups of mammals. However, we also show that many species lack TRIM5 and TRIM22 altogether. By analyzing a large number of mammalian genomes, here we provide the first comprehensive view of the evolution of these genes in eutherians, showcasing that the pattern of accumulation of TRIM genes has been dissimilar across mammalian orders. Our data suggest that these differences are caused by the evolutionary plasticity of the immune system of eutherians, which have adapted to use different strategies to combat retrovirus infections. Altogether, our results provide insights into the dissimilar evolution of a representative family of restriction factors, highlighting an example of adaptive and idiosyncratic evolution in the innate immune system.