Wentao Qiao

Foamy viruses (FVs) are capable of cross-species transmission to humans and establish latent infections without causing diseases. The transition between latent and lytic FV replication is regulated by the viral proteins Bet and Tas, which are expressed predominantly from the internal promoter (IP). Bet suppresses basal IP activity and initiates latency, whereas Tas activates the IP and the long terminal repeat (LTR) promoter and enables lytic infection. However, the mechanisms regulating the relative levels of Tas and Bet are not completely understood. We performed a yeast two-hybrid screen to identify Tas-interacting proteins and discovered an association of the splicing factor heterogeneous nuclear ribonucleoprotein F (hnRNPF) with Tas. We found that hnRNPF inhibits prototype foamy virus (PFV) replication by shifting the viral expression profile from Tas to Bet. Importantly, hnRNPF specifically recognizes a G-cluster II cis-acting element (GC-II) within the tas/bet pre-mRNA to promote its splicing into bet mRNA, thereby reducing the levels of the unspliced tas transcript. The viral Tas protein, which interacts with hnRNPF, also possesses this splicing-promoting activity. Tas acts by binding to its DNA target, the Tas responsive element (TRE), thereby recruiting the hnRNPF to nascent viral RNA to enhance splicing efficiency. We conclude that PFV Tas regulates the expression of itself and the viral Bet protein through splicing of tas/bet pre-mRNA by recruiting the cellular splicing factor hnRNPF. We further propose that hnRNPF may play an important role in establishing PFV latency by assisting Tas in decreasing Tas levels and increasing Bet expression to shut down viral gene expression.

Oncolytic viruses represent an innovative strategy for cancer therapy. However, extensive gene expression reprogramming within tumor cells may hinder viral propagation by affecting essential cell-virus interactions. Here, through genome-wide CRISPR/Cas9 library screening, Suppressor of variegation 3–9 homolog 2 (SUV39H2), a histone methyltransferase, was identified as a critical factor in mediating resistance to oncolytic herpes simplex virus 1 (oHSV-1) in oral squamous cell carcinoma (OSCC). Functional studies in SCC15 cells revealed that SUV39H2 knockdown facilitated viral replication, while its overexpression suppressed it. The inhibitor OTS186935 targeting SUV39H2 was administered to evaluate its effects on viral replication both in vitro and in vivo. Pretreatment with OTS186935 in SCC15, SCC7, and MCF7 led to a significant enhancement of viral replication. Combined treatment with OTS186935 and oHSV-1 demonstrated significant anti-tumor efficacy in BALB/c nude mice bearing SCC15 tumors. SUV39H2 was shown to regulate the trimethylation of lysine 9 on histone 3 (H3K9me3) at the viral promoter regions of immediate-early gene ICP0, ICP4 and early gene ICP8, thereby repressed viral gene transcription. However, oHSV-1 infection induced the degradation of SUV39H2, a process mediated by the viral protein ICP0 through the proteasomal pathway. Findings from studies in SCC7 cells further supported the observation that SUV39H2 knockdown enhanced viral replication. Moreover, SUV39H2 downregulation increased CD4+ and CD8+ T cell infiltration in syngeneic tumors treated with oHSV-1. TCGA database analysis revealed that SUV39H2 is associated with distinct immune cell infiltration patterns across different cancer types and correlates with immune checkpoint expression. These results highlight the role of SUV39H2 in regulating oHSV-1 replication and indicate that SUV39H2 may represent a potential target to improve the efficacy of oncolytic virotherapy.

Prototype foamy viruses (PFVs) are complex retroviruses that establish long-term latent infections in hosts without causing disease, positioning them as potential safe gene transfer vectors. Understanding the host proteins involved in PFV replication and their interaction mechanisms may enhance gene transfer efficiency. However, only a few cellular proteins are known to influence PFV replication. In this study, transcriptomic analysis of PFV-infected HT1080 cells revealed significant downregulation of variable charge Y (VCY) mRNA, indicating its potential significance in regulating PFV replication. Overexpression of VCY significantly inhibits PFV replication, whereas VCY knockdown enhances viral replication, which can be reversed by reintroducing the VCY protein. VCY interacted with the Tas DNA-binding and transcriptional activation domains and interfered with its binding to the PFV long terminal repeat and internal promoter, inhibiting the transactivation function of Tas. The N-terminal region of VCY, which contains a nuclear localization signal, is essential for this function. Additionally, VCY suppresses bovine foamy virus (BFV) replication by impairing the transactivation activity of BFV Tas, suggesting a broad-spectrum inhibitory effect on FV replication. Collectively, our data elucidate the role of VCY in inhibiting PFV replication through transcriptional interference for the first time, providing valuable insights into viral latency and host interactions. IMPORTANCE: FVs can integrate into host chromosomes and are nonpathogenic in natural hosts or experimentally infected animals, making them safe and efficient gene transfer vectors. They establish lifelong latent infections without evident pathology in the host. To date, only a few host factors have been identified that affect PFV replication. In this study, we report that VCY inhibits PFV replication by modulating the function of the transcription activator Tas. Currently, there have been no studies examining the relationship between VCY and viruses, making this the inaugural report on its association with viral infection. Our data provide important insights into the role of VCY in PFV life cycle, which will aid in understanding the mechanisms underlying retroviral latent infection.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles. IMPORTANCE: In this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors.

CRF01_AE strains have been shown to form multiple transmission clusters in China, and some clusters have disparate pathogenicity in Chinese men who have sex with men. This study focused on other CRF01_AE clusters prevalent in heterosexual populations. The CD4 ⁺ T-cell counts from both cross-section data in National HIV Molecular Epidemiology Survey and seropositive cohort data were used to evaluate the pathogenicity of the CRF01_AE clusters and other HIV-1 sub-types. Their mechanisms of pathogenicity were evaluated by co-receptor tropisms, predicted by genotyping and confirmed with virus isolate phenotyping, as well as inflammation parameters. Our research elucidated that individuals infected with CRF01_AE clusters 1 and 2 exhibited significantly lower baseline CD4 ⁺ T-cell counts and greater CD4 ⁺ T-cell loss in cohort follow-up, compared with other HIV-1 sub-types and CRF01_AE clusters. The increased pathogenesis of cluster 1 or 2 was associated with higher CXCR4 tropisms, higher inflammation/immune activation, and increased pyroptosis. The protein structure modeling analysis revealed that the envelope V3 loop of clusters 1 and 2 viruses is favorable for CXCR4 co-receptor usage. Imbedded with the most mutating reverse transcriptase, HIV-1 is one of the most variable viruses. CRF01_AE clusters 1 and 2 have been found to have evolved into more virulent strains in regions with predominant heterosexual infections. The virulent strains increased the pressure for early diagnosis and treatment in HIV patients. To save more lives, HIV-1 surveillance systems should be upgraded from serology and genotyping to phenotyping, which could support precision interventions for those infected by virulent viruses. IMPORTANCE: Retroviruses swiftly adapt, employing error-prone enzymes for genetic and phenotypic evolution, optimizing survival strategies, and enhancing virulence levels. HIV-1 CRF01_AE has persistently undergone adaptive selection, and cluster 1 and 2 infections display lower counts and fast loss of CD4 ⁺ T cells than other HIV-1 sub-types and CRF01_AE clusters. Its mechanisms are associated with increased CXCR4 tropism due to an envelope structure change favoring a tropism shift from CCR5 to CXCR4, thereby shaping viral phenotype features and impacting pathogenicity. This underscores the significance of consistently monitoring HIV-1 genetic evolution and phenotypic transfer to see whether selection bias across risk groups alters the delicate balance of transmissible versus toxic trade-offs, since virulent strains such as CRF01_AE clusters 1 and 2 could seriously compromise the efficacy of antiviral treatment. Only through such early warning and diagnostic services can precise antiviral treatments be administered to those infected with more virulent HIV-1 strains.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for the budding of retroviruses such as human immunodeficiency virus (HIV) and bovine foamy virus (BFV), which rely on their late domain to recruit ESCRT complexes to facilitate budding. However, the impact of intracellular host proteins on BFV budding remains poorly understood. In this study, we aimed to investigate the impact of CCL2 on BFV budding and interactions with key host proteins. Our results indicate that CCL2 promotes BFV budding in an ALG-2-interacting protein X (Alix)-dependent manner by enhancing the interaction between Alix and BFV Gag (BGag). Notably, we found a link between Alix, BGag and CCL2, with Alix mediating the interaction between the latter two. Furthermore, we observed that natural host bovine CCL2 also has a facilitating role in the budding process of BFV, similar to human CCL2. Taken together, these results demonstrate that CCL2 promotes BFV budding by enhancing the Alix-BGag association.

Background: Foamy viruses (FVs) are retroviruses with unique replication strategies that cause lifelong latent infections in their hosts. FVs can also produce foam-like cytopathic effects in vitro. However, the effect of host cytokines on FV replication requires further investigation. Although interferon induced transmembrane (IFITMs) proteins have become the focus of antiviral immune response research due to their broad-spectrum antiviral ability, it remains unclear whether IFITMs can affect FV replication. Method: In this study, the PFV virus titer was characterized by measuring luciferase activity after co-incubation of PFVL cell lines with the cell culture supernatants (cell-free PFV) or the cells transfected with pcPFV plasmid/infected with PFV (cell-associated PFV). The foam-like cytopathic effects of PFV infected cells was observed to reflect the virus replication. The total RNA of PFV infected cells was extracted, and the viral genome was quantified by Quantitative reverse transcription PCR to detect the PFV entry into target cells. Results: In the present study, we demonstrated that IFITM1-3 overexpression inhibited prototype foamy virus (PFV) replication. In addition, an IFITM3 knockdown by small interfering RNA increased PFV replication. We further demonstrated that IFITM3 inhibited PFV entry into host cells. Moreover, IFITM3 also reduced the number of PFV envelope proteins, which was related to IFITM3 promoted envelope degradation through the lysosomal pathway. Conclusions: Taken together, these results demonstrate that IFITM3 inhibits PFV replication by inhibiting PFV entry into target cells and reducing the number of PFV envelope.