The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor α (HIF-1α) during latent infection, and HIF-1α reactivates lytic replication under hypoxic stress. However, the mechanism utilized by KSHV to block lytic reactivation with the accumulation of HIF-1α in latency remains unclear. Here, we report that LANA encoded by KSHV contains a unique SUMO-interacting motif (LANA^SIM) which is specific for interaction with SUMO-2 and facilitates LANA SUMOylation at lysine 1140. Proteomic and co-immunoprecipitation analysis further reveal that the SUMO-2 modified transcription repressor KAP1 is a critical factor recruited by LANA^SIM. Deletion of LANA^SIM led to functional loss of both LANA-mediated viral episome maintenance and lytic gene silencing. Moreover, hypoxia reduced KAP1 SUMOylation and resulted in dissociation of both KAP1 and Sin3A repressors from LANA^SIM-associated complex. Therefore, the LANA^SIM motif plays an essential role in KSHV latency and is a potential drug target against KSHV-associated cancers. Hypoxia stress is a common feature of tumor microenvironment and is widely associated with pathogenesis linked to many oncogenic viruses. Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological cause of Kaposi's sarcoma and primary effusion lymphoma, has been reported to encode several proteins that usurp hypoxia signaling during infection. One encoded protein LANA is a latent protein important for regulation of KSHV life cycle and cell transformation. The molecular mechanisms of how KSHV controls life cycle in normoxia and hypoxia is not fully understood. In this study, we show that LANA contains a unique SUMO-interacting motif (LANA^SIM) which is specific for SUMO-2 binding. Importantly, SUMO-2 modified KAP1, a chromatin remodeling factor recruited by LANA^SIM is hypoxia sensitive, and plays a critical role in silencing viral gene expression. This discovery not only adds to our understanding of hypoxia-mediated remodeling of the viral episome in a SUMO dependent manner, but also provides a new dimension to development of therapeutic strategies against KSHV-associated cancers.

Lung cancer (LC) is a leading cause of cancer-related deaths worldwide. Its rapid growth requires hyperactive catabolism of principal metabolic fuels. It is unclear whether fructose, an abundant sugar in current diets, is essential for LC. We demonstrated that, under the condition of coexistence of metabolic fuels in the body, fructose was readily used by LC cells in vivo as a glucose alternative via upregulating GLUT5, a major fructose transporter encoded by solute carrier family 2 member 5 ( SLC2A5 ). Metabolomic profiling coupled with isotope tracing demonstrated that incorporated fructose was catabolized to fuel fatty acid synthesis and palmitoleic acid generation in particular to expedite LC growth in vivo. Both in vitro and in vivo supplement of palmitoleic acid could restore impaired LC propagation caused by SLC2A5 deletion. Furthermore, molecular mechanism investigation revealed that GLUT5-mediated fructose utilization was required to suppress AMPK and consequently activate mTORC1 activity to promote LC growth. As such, pharmacological blockade of in vivo fructose utilization using a GLUT5 inhibitor remarkably curtailed LC growth. Together, this study underscores the importance of in vivo fructose utilization mediated by GLUT5 in governing LC growth and highlights a promising strategy to treat LC by targeting GLUT5 to eliminate those fructose-addicted neoplastic cells.

Journal of Medical Internet Research - International Scientific Journal for Medical Research, Information and Communication on the Internet #Preprint #PeerReviewMe: Warning: This is a unreviewed preprint. Readers are warned that the document has not been peer-reviewed by expert/patient reviewers or an academic editor, may contain misleading claims, and is likely to undergo changes before final publication, if accepted, or may have been rejected/withdrawn. Readers with interest and expertise are encouraged to sign up as peer-reviewer, if the paper is within an open peer-review period. Please cite this preprint only for review purposes or for grant applications and CVs (if you are the author). Background: Cancers pose a serious threat to health of Chinese people, resulting in a immense challenge to public health work. Nowadays, people could get related information not only from medical workers in hospitals, but also retrieve on Internet everywhere in real-time. Online search behaviors can reflect population’s awareness on cancers from a completely new perspective, which could be driven by underlying cancer epidemiology. Objective: The objective of our study was mainly to explore whether there was a correlation among incidence and mortaliy of cancers and online search behaviors. A secondary objective was to predict future incidence and mortality of theses cancers if the correlation was existed. Methods: We used standard Boolean operators to choose search terms of each cancer. We used Spearman’s rank correlation coefficient to validate the correlation among monthly search index values of each cancer type and their monthly incidence and mortality rates. ARIMA model and linear regression model were simultaneously used to predict incidence and mortality of these cancers in future. Results: Monthly search index values statistically significantly correlated with cancer incidence rates in 26 of 28 cancers in China, ( lung cancer: r=.80, P<.001; liver cancer: r=.28, P=.016; stomach cancer: r=.50, P<.001; esophageal cancer: r=.50, P<.001; colorectal cancer: r=.81, P<.001; pancreatic cancer: r=.86, P<.001; breast cancer: r=.56, P<.001; brain and nervous system cancer: r=.63, P<.001; leukemia: r=.75, P<.001; Non-Hodgkin lymphoma: r=.88, P<.001; Hodgkin lymphoma: r=.91, P<.001; cervical cancer: r=.64, P<.001; prostate cancer: r=.67, P<.001; bladder cancer: r=.62, P<.001; gallbladder and biliary tract cancer: r=.88, P<.001; lip and oral cavity cancer: r=.88, P<.001; ovarian cancer: r=.58, P<.001; larynx cancer: r=.82, P<.001; kidney cancer: r=.73, P<.001; squamous-cell carcinoma: r=.94, P<.001; multiple myeloma: r=.84, P<.001; thyroid cancer: r=.77, P<.001; malignant skin melanoma: r=.55, P<.001; mesothelioma: r=.79, P<.001; testicular cancer: r=.57, P<.001; basal-cell carcinoma: r=.83, P<.001). Meanwhile, monthly Baidu search index values statistically significantly correlated with cancer mortality rates in 24 of 27 cancers but not for stomach cancer, esophageal cancer and testicular cancer. Conclusions: Online search behaviors indeed reflect public awareness from a different angle. Research on Internet search behaviors may be an innovative and advance-time tool to monitor and estimate cancer incidence and mortality rates, especially for cancers not included in national registries.

Hypoxia-inducible factor 1α (HIF-1α) has been frequently implicated in many cancers as well as viral pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. It can stabilize HIF-1α during latent infection and undergoes lytic replication in response to hypoxic stress. However, the mechanism by which KSHV controls its latent and lytic life cycle through the deregulation of HIF-1α is not fully understood. Our previous studies showed that the hypoxia-sensitive chromatin remodeler KAP1 was targeted by the KSHV-encoded latency-associated nuclear antigen (LANA) to repress expression of the major lytic replication and transcriptional activator (RTA). Here we further report that an RNA interference-based knockdown of KAP1 in KSHV-infected primary effusion lymphoma (PEL) cells disrupted viral episome stability and abrogated sub-G ₁ /G ₁ arrest of the cell cycle while increasing the efficiency of KSHV lytic reactivation by hypoxia or using the chemical 12- O -tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate (NaB). Moreover, KSHV genome-wide screening revealed that four hypoxia-responsive clusters have a high concurrence of both RBP-Jκ and HIF-1α binding sites (RBS+HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-Jκ with the HIF-1α complex for driving RTA expression not only in normoxia but also in hypoxia. These results suggest that both KAP1 and the concurrence of RBS+HRE within the RTA promoter are essential for KSHV latency and hypoxia-induced lytic reactivation. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV), a DNA tumor virus, is an etiological agent linked to several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). HIF-1α, a key hypoxia-inducible factor, is frequently elevated in KSHV latently infected tumor cells and contributes to KSHV lytic replication in hypoxia. The molecular mechanisms of how KSHV controls the latent and lytic life cycle through deregulating HIF-1α remain unclear. In this study, we found that inhibition of hypoxia-sensitive chromatin remodeler KAP1 in KSHV-infected PEL cells leads to a loss of viral genome and increases its sensitivity to hypoxic stress, leading to KSHV lytic reactivation. Importantly, we also found that four hypoxia-responsive clusters within the KSHV genome contain a high concurrence of RBP-Jκ (a key cellular regulator involved in Notch signaling) and HIF-1α binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-Jκ, and HIF-1α play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.

Manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment during infection. Kaposi’s sarcoma-associated herpesvirus (KSHV), the primary etiological agent of several human malignancies including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), encodes several oncoproteins that deregulate normal physiology of cell cycle machinery to persist with endothelial cells and B cells and subsequently establish a latent infection. During latency, only a small subset of viral proteins is expressed. LANA (Latency-associated nuclear antigen) is one of the latent antigens shown to be essential for transformation of endothelial cells in vitro. It has been well demonstrated that LANA is critical for the maintenance of latency, episome DNA replication, segregation and gene transcription. In this review, we summarize recent studies and address how LANA functions as an oncoprotein to steer host cell cycle-related events including proliferation and apoptosis by interacting with various cellular and viral factors, and highlight the potential therapeutic strategy of disrupting LANA-dependent signaling as targets in KSHV-associated cancers.