A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Development and Evaluation of an Ebola Virus Glycoprotein Mucin-Like Domain Replacement System as a New Dendritic Cell-Targeting Vaccine Approach against HIV-1.

The development of efficient vaccine approaches against HIV infection remains challenging in the vaccine field. Here, we developed an Ebola virus envelope glycoprotein (EboGP)-based chimeric fusion protein system and demonstrated that replacement of the mucin-like domain (MLD) of EboGP with HIV C2-V3-C3 (134 amino acids [aa]) or C2-V3-C3-V4-C4-V5-C5 (243 aa) polypeptides (EbGPΔM-V3 and EbGPΔM-V3-V5, respectively) still maintained the efficiency of EboGP-mediated viral entry into human macrophages and dendritic cells (DCs). Animal studies using mice revealed that immunization with virus-like particles (VLPs) containing the above chimeric proteins, especially EbGPΔM-V3, induced significantly more potent anti-HIV antibodies than HIV gp120 alone in mouse serum and vaginal fluid. Moreover, the splenocytes isolated from mice immunized with VLPs containing EbGPΔM-V3 produced significantly higher levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-4, IL-5, and macrophage inflammatory protein 1α (MIP-1α). Additionally, we demonstrated that coexpression of EbGPΔM-V3 and the HIV Env glycoprotein in a recombinant vesicular stomatitis virus (rVSV) vector elicited robust anti-HIV antibodies that may have specifically recognized epitopes outside or inside the C2-V3-C3 region of HIV-1 gp120 and cross-reacted with the gp120 from different HIV strains. Thus, this study has demonstrated the great potential of this DC-targeting vaccine platform as a new vaccine approach for improving immunogen delivery and increasing vaccine efficacy. IMPORTANCE Currently, there are more than 38.5 million reported cases of HIV globally. To date, there is no approved vaccine for HIV-1 infection. Thus, the development of an effective vaccine against HIV infection remains a global priority. This study revealed the efficacy of a novel dendritic cell (DC)-targeting vaccination approach against HIV-1. The results clearly show that the immunization of mice with virus-like particles (VLPs) and VSVs containing HIV Env and a fusion protein composed of a DC-targeting domain of Ebola virus GP with HIV C2-V3-C3 polypeptides (EbGPΔM-V3) could induce robust immune responses against HIV-1 Env and/or Gag in serum and vaginal mucosa. These findings provide a proof of concept of this novel and efficient DC-targeting vaccine approach in delivering various antigenic polypeptides of HIV-1 and/or other emergent infections to the host antigen-presenting cells to prevent HIV and other viral infections.

Noncovalent SUMO-interaction motifs in HIV integrase play important roles in SUMOylation, cofactor binding, and virus replication.

BACKGROUND: HIV integrase (IN) and its cellular cofactors, including lens-epithelium-derived growth factor (LEDGF/p75), Ku70, p300, and Rad52, are subject to small ubiquitin-like modifier (SUMO) modification. In addition to covalent SUMOylation, SUMO paralogs can also noncovalently bind proteins through SUMO-interacting motifs (SIMs). However, little is known about whether HIV IN contains SIMs and the roles of these motifs. RESULTS: We searched for the amino acid sequence of HIV IN and investigated three putative SIMs of IN: SIM1 72VILV75, SIM2 200IVDI203 and SIM3 257IKVV260. Our mutational analysis showed that 200IVDI203 and 257IKVV260 are two bona fide SIMs that mediate IN-SUMO noncovalent interactions. Additionally, a cell-based SUMOylation assay revealed that IN SIMs negatively regulate the SUMOylation of IN, as well as the interaction between IN and SUMO E2 conjugation enzyme Ubc9. Conversely, IN SIMs are required for its interactions with LEDGF/p75 but not with Ku70. Furthermore, our study reveals that SIM2 and SIM3 are required for the nuclear localization of IN. Finally, we investigated the impact of IN SIM2 and SIM3 on HIV single cycle replication in CD4+ C8166 T cells, and the results showed that viruses carrying IN SIM mutants are replication defective at the steps of the early viral life cycle, including reverse transcription, nuclear import and integration. CONCLUSION: Our data suggested that the INSIM-SUMO interaction constitutes a new regulatory mechanism of IN functions and might be important for HIV-1 replication.

Human immunodeficiency virus type 1 employs the cellular dynein light chain 1 protein for reverse transcription through interaction with its integrase protein.

UNLABELLED: In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150(Glued) in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150(Glued), resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs (52)GQVD and (250)VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1IN(Q53A/Q252A)) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1IN(Q53A/Q252A) mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE: Host cellular DYNLL1, DYNLT1, and p150(Glued) proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral replication steps.

Activation of HIV-1 expression in latently infected CD4+ T cells by the small molecule PKC412.

BACKGROUND: HIV-1 latency is a major obstacle for HIV-1 eradication. Extensive efforts are being directed toward the reactivation of latent HIV reservoirs with the aim of eliminating latently infected cells via the host immune system and/or virus-mediated cell lysis. RESULTS: We screened over 1,500 small molecules and kinase inhibitors and found that a small molecule, PKC412 (midostaurin, a broad-spectrum kinase inhibitor), can stimulate viral transcription and expression from the HIV-1 latently infected ACH2 cell line and primary resting CD4+ T cells. PKC412 reactivated HIV-1 expression in ACH2 cells in a dose- and time-dependent manner. Our results also suggest that the nuclear factor κB (NF-κB) signaling could be one of cellular pathways activated during PKC412-mediated activation of latent HIV-1 expression. Additionally, combining PKC412 with the HDAC inhibitor vorinostat (VOR) had an additive effect on HIV-1 reactivation in both ACH2 cells and infected resting CD4+ T cells. CONCLUSIONS: These studies provide evidence that PKC412 is a new compound with the potential for optimization as a latency-reactivator to eradicate HIV-1 infection.

UV-Inactivated rVSV-M2e-Based Influenza Vaccine Protected against the H1N1 Influenza Challenge.

BACKGROUND: To investigate the immune responses and protection ability of ultraviolet light (UV)-inactivated recombinant vesicular stomatitis (rVSV)-based vectors that expressed a fusion protein consisting of four copies of the influenza matrix 2 protein ectodomain (tM2e) and the Dendritic Cell (DC)-targeting domain of the Ebola Glycoprotein (EΔM), (rVSV-EΔM-tM2e). METHOD: In our previous study, we demonstrated the effectiveness of rVSV-EΔM-tM2e to induce robust immune responses against influenza M2e and protect against lethal challenges from H1N1 and H3N2 strains. Here, we used UV to inactivate rVSV-EΔM-tM2e and tested its immunogenicity and protection in BALB/c mice from a mouse-adapted H1N1 influenza challenge. Using Enzyme-Linked Immunosorbent Assay (ELISA) and Antibody-Dependent Cellular Cytotoxicity (ADCC), the influenza anti-M2e immune responses specific to human, avian and swine influenza strains induced were characterized. Likewise, the specificity of the anti-M2e immune responses induced in recognizing M2e antigen on the surface of the cell was investigated using Fluorescence-Activated Cell Sorting (FACS) analysis. RESULTS: Like the live attenuated rVSV-EΔM-tM2e, the UV-inactivated rVSV-EΔM-tM2e was highly immunogenic against different influenza M2e from strains of different hosts, including human, swine, and avian, and protected against influenza H1N1 challenge in mice. The FACS analysis demonstrated that the induced immune responses can recognize influenza M2 antigens from human, swine and avian influenza strains. Moreover, the rVSV-EΔM-tM2e also induced ADCC activity against influenza M2e from different host strains. CONCLUSIONS: These findings suggest that UV-inactivated rVSV-EΔM-tM2e could be used as an inactivated vaccine against influenza viruses.

Contribution of host nucleoporin 62 in HIV-1 integrase chromatin association and viral DNA integration.

HIV-1 integration is promoted by viral integrase (IN) and its cellular cofactors. The lens epithelium-derived growth factor (LEDGF/p75), an IN interacting cellular cofactor, has been shown to play an important role in HIV-1 chromatin targeting and integration. However, whether other cellular cofactors are also involved in viral replication steps is still elusive. Here, we show that nucleoporin 62 (Nup62) is a chromatin-bound protein and can specifically interact with HIV-1 IN in both soluble nuclear extract and chromatin-bound fractions. The knockdown of Nup62 by shRNA reduced the association of IN with host chromatin and significantly impaired viral integration and replication in HIV-1-susceptible cells. Furthermore, the expression of the IN-binding region of Nup62 in CD4(+) T cells significantly inhibited HIV-1 infection. Taken together, these results indicate that the cellular Nup62 is specifically recruited by HIV-1 IN and contribute to an efficient viral DNA integration.

Characterization of antiviral activity of benzamide derivative AH0109 against HIV-1 infection.

In the absence of an effective vaccine against HIV-1 infection, anti-HIV-1 strategies play a major role in disease control. However, the rapid emergence of drug resistance against all currently used anti-HIV-1 molecules necessitates the development of new antiviral molecules and/or strategies against HIV-1 infection. In this study, we have identified a benzamide derivative named AH0109 that exhibits potent anti-HIV-1 activity at an 50% effective concentration of 0.7 µM in HIV-1-susceptible CD4(+) C8166 T cells. Mechanistic analysis revealed that AH0109 significantly inhibits both HIV-1 reverse transcription and viral cDNA nuclear import. Furthermore, our infection experiments indicated that AH0109 is capable of disrupting the replication of HIV-1 strains that are resistant to the routinely used anti-HIV-1 drugs zidovudine, lamivudine, nevirapine, and raltegravir. Together, these findings provide evidence for a newly identified antiviral molecule that can potentially be developed as an anti-HIV-1 agent.

The cellular antiviral protein APOBEC3G interacts with HIV-1 reverse transcriptase and inhibits its function during viral replication.

The cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editing-mediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G(65-132) was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription.

[Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells].

OBJECTIVE: To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell. METHODS: Firstly (Lenti-GFP-CGRP, referred to CGRP (+/+)) MSCs were transfected with high expression lentivirus vector of CGRP (MSCs(CGRP+/+)). Protein secretion in the above-mentioned MSCs(CGRP+/+) supernatant was detected with enzyme-linked immunosorbent assay (ELISA). And then MSCs(CGRP+/+) was co-cultured with VSMCs(RAMP1)(+/+) and VSMCs(RAMP1-/-) respectively. Experimental groups were as follows: MSCs +VSMCs MSCs(CGRP+/+) +VSMCs, MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+) and MSCs(CGRP+/+) + VSMCs (RAMP1-/-). Flow cytometry was applied to detect the cycle variation of smooth muscle cells, thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively. RESULTS: After the transfection of CGRP(+/+), MSCs(CGRP+/+) secreted and expressed CGRP protein, the secretory volume of CGRP protein in MSCs(CGRP+/+) increased significantly compared with the control group (19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001, P < 0.05) . After a 72 h co-culturing with VSMCs, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+)group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070, P < 0.05) and the number of cells staying in G0 phase significantly increased (93.51% ± 0.38% vs 84.48% ± 0.31%, P < 0.05) , the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP(+/+) +VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ± 635, P < 0.05), OPN (26 026 ± 2595 vs 44 201 ± 2811, P < 0.05) }; but compared with MSCs(CGRP+/+)+VSMCs(RAMP1)(+/+)group, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)-/-group significantly increased (0.601 ± 0.04 vs 0.270 ± 0.263, P < 0.05) while the number of cells staying in G0 phase significantly declined (78.57% ± 0.68% vs 93.51% ± 0.38%, P < 0.05) . The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCs(CGRP+/+)+VSMCs(RAMP1)-/-group {α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847, P < 0.05), OPN (53 933 ± 1192 vs 26 026 ± 2595, P < 0.05)}. CONCLUSIONS: CGRP gene-modified MSCs may secrete target protein stably. And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation. It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.

Protocol to evaluate the inflammatory response in human macrophages induced by SARS-CoV-2 spike-pseudotyped VLPs.

The excessive release of pro-inflammatory cytokines in COVID-19 patients is deleterious to organs. The contribution of SARS-CoV-2 spike protein (S) to the inflammatory response is essential to understand its pathogenesis and virulence. Here, we present a protocol to produce and characterize HIV- and SARS-CoV-2-based virus-like particles and then evaluate the inflammatory cytokines' protein and mRNA levels produced in human macrophages by S of SARS-CoV-2 original strain and Delta variant. This protocol is applicable in evaluating S from different emerging variants. For complete details on the use and execution of this protocol, please refer to Ao et al. (2022).1.

Significance of Conserved Regions in Coronavirus Spike Protein for Developing a Novel Vaccine against SARS-CoV-2 Infection.

Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2, which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants' proteins, especially in the spike protein (SP) used for viral entry. These mutations, especially in the SP, enable the virus to evade immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of a vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase its immunogenicity.

Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, against Both SARS-CoV-2 and Influenza A Viruses.

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Host protein Ku70 binds and protects HIV-1 integrase from proteasomal degradation and is required for HIV replication.

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys(48)-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230-288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1-430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4(+) T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.

SARS-CoV-2 Delta spike protein enhances the viral fusogenicity and inflammatory cytokine production.

The Delta variant had spread globally in 2021 and caused more serious disease than the original virus and Omicron variant. In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation, its impact on viral entry of pseudovirus and cell-cell fusion, and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SPΔCDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). Further, SPΔCDelta elevated pseudovirus entry in human lung cell lines and significantly enhanced syncytia formation. Furthermore, we revealed that SPΔCDelta-PVLPs had stronger effects on stimulating NF-κB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of proinflammatory cytokine, such as TNF-α, IL-1ß, and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SPΔCDelta during virus infection, transmission, and pathogenesis.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Importin alpha3 interacts with HIV-1 integrase and contributes to HIV-1 nuclear import and replication.

HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin alpha (Impalpha) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Impalpha3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4(+) C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Impalpha3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Impalpha3 in HIV nuclear import. In an attempt to understand how Impalpha3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Impalpha3 both in a 293T cell expression system and in HIV-infected CD4(+) C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Impalpha3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.

Identification and evaluation of the inhibitory effect of Prunella vulgaris extract on SARS-coronavirus 2 virus entry.

Until now, antiviral therapeutic agents are still urgently required for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a sensitive SCoV-2 Spike glycoprotein (SP), including an SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. Based on this system, we have demonstrated that an aqueous extract from the Natural herb Prunella vulgaris (NhPV) displayed potent inhibitory effects on SCoV-2 SP (including SPG614 mutant) pseudotyped virus (SCoV-2-SP-PVs) mediated infections. Moreover, we have compared NhPV with another compound, Suramin, for their anti-SARS-CoV-2 activities and the mode of their actions, and found that both NhPV and Suramin are able to directly interrupt SCoV-2-SP binding to its receptor ACE2 and block the viral entry step. Importantly, the inhibitory effects of NhPV and Suramin were confirmed by the wild type SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) virus infection in Vero cells. Furthermore, our results also demonstrated that the combination of NhPV/Suramin with an anti-SARS-CoV-2 neutralizing antibody mediated a more potent blocking effect against SCoV2-SP-PVs. Overall, by using SARS-CoV-2 SP-pseudotyped HIV-1-based entry system, we provide strong evidence that NhPV and Suramin have anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.

Characterization of the HIV-1 integrase chromatin- and LEDGF/p75-binding abilities by mutagenic analysis within the catalytic core domain of integrase.

BACKGROUND: During the early stage of HIV-1 replication, integrase (IN) plays important roles at several steps, including reverse transcription, viral DNA nuclear import, targeting viral DNA to host chromatin and integration. Previous studies have demonstrated that HIV-1 IN interacts with a cellular Lens epithelium-derived growth factor (LEDGF/p75) and that this viral/cellular interaction plays an important role for tethering HIV-1 preintegration complexes (PICs) to transcriptionally active units of host chromatin. Meanwhile, other studies have revealed that the efficient knockdown and/or knockout of LEDGF/p75 could not abolish HIV infection, suggesting a LEDGF/p75-independent action of IN for viral DNA chromatin targeting and integration, even though the underlying mechanism(s) is not fully understood. RESULTS: In this study, we performed site-directed mutagenic analysis at the C-terminal region of the IN catalytic core domain responsible for IN/chromatin binding and IN/LEDGF/p75 interaction. The results showed that the IN mutations H171A, L172A and EH170,1AA, located in the loop region 170EHLK173 between the alpha4 and alpha5 helices of IN, severely impaired the interaction with LEDGF/p75 but were still able to bind chromatin. In addition, our combined knockdown approach for LEDGF/p75 also failed to dissociate IN from chromatin. This suggests that IN has a LEDGF/p75-independent determinant for host chromatin binding. Furthermore, a single-round HIV-1 replication assay showed that the viruses harboring IN mutants capable of LEDGF/p75-independent chromatin binding still sustained a low level of infection, while the chromatin-binding defective mutant was non-infectious. CONCLUSIONS: All of these data indicate that, even though the presence of LEDGF/p75 is important for a productive HIV-1 replication, IN has the ability to bind chromatin in a LEDGF/p75-independent manner and sustains a low level of HIV-1 infection. Hence, it is interesting to define the mechanism(s) underlying IN-mediated LEDGF/p75-independent chromatin targeting, and further studies in this regard will help for a better understanding of the molecular mechanism of chromatin targeting by IN during HIV-1 infection.

Reduced HIV-1 long terminal repeat transcription in subjects with protective interferon regulatory factor-1 genotype: a potential mechanism mediating resistance to infection by HIV-1.

We previously described the polymorphism in the interferon regulatory factor-1 (IRF-1) gene as a novel correlate of resistance to HIV-1 infection in a Kenyan female sex worker cohort. However, the underlying mechanisms likely mediating this association remained to be elucidated. The initiation of HIV-1 long terminal repeat (LTR) transcription in peripheral blood mononuclear cells (PBMCs) from subjects with different IRF-1 haplotypes, representing protective, intermediate and the least protective IRF-1 allele combinations, were investigated here. A single-cycle pseudovirus construct expressing vesicular stomatitis virus envelop G-protein (VSV-G) and having an HIV-1 pNL4.3 backbone with luciferase insert was used to infect PBMCs with different IRF-1 haplotypes. The efficiency of early HIV-1 LTR transcription was monitored using a luciferase assay. IRF-1 protein levels induced by the infection were measured by quantitative Western blot. Our results showed that PBMCs with the protective IRF-1 genotype demonstrated significantly lower HIV-1 LTR transcription during the initial stages of infection compared to PBMCs with other haplotypes, which correlated with the kinetics of IRF-1 responsiveness to HIV-1 infection in the cells. It suggests that IRF-1 genotypes alter the efficiency of early HIV-1 LTR transcription, likely via modulating expression of IRF-1. This may represent one mechanism mediating the association between IRF-1 polymorphisms and resistance to HIV-1 infection.