Infectivity Of Progeny Virions Research Articles

Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle

ABSTRACT Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

DNAJB8 facilitates autophagic-lysosomal degradation of viral Vif protein and restricts HIV-1 virion infectivity by rescuing APOBEC3G expression in host cells.

HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell–cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell–cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

SNAP25 regulates the release of the Rabies virus in nerve cells via SNARE complex-mediated membrane fusion

Recent studies have reported that host proteins regulate Rabies virus (RABV) infection via distinct mechanisms. The abnormal neural function caused by RABV infection is related to the abnormal synaptic signal transmission in which the RABV glycoprotein (G) is involved. In the present study, two recombinant Rabies viruses (rRABVs), namely rSAD-SAD-Flag-G and rSAD-CVS-Flag-G, were established and rescued based on rSAD and verified by indirect fluorescence assay (IFA), and western blotting (WB). To investigate how the G protein interacts with synaptosomal-associated protein 25 (SNAP25), primary neuronal cells (PNC) of embryonic mice were cultured and infected with rRABVs. Immunoprecipitation (IP) and LC–MS/MS analysis of glycoprotein-binding proteins, which were flag tagged, were carried out to determine the interaction of G protein and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNARE) complex in PNC. G protein and the SNARE member SNAP25 were co-expressed in HEK293 cells or primary neuronal cells to investigate their colocalization. Knockdown of SNAP25 with small interfering RNA (siRNA) was conducted on mNA cells, and rRABV replication was observed by IFA, qRT-PCR, and virus titration. The results indicated that rRABVs were successfully rescued and grew well in PNC. Flag-tag IP and confocal microscopy demonstrated that SNAP25 works together with G protein and colocalizes with G on the cytomembrane of HEK293 cells. The downregulation of SNAP25, using RNA interference, resulted in a significant decrease in the number of viral mRNAs, viral proteins, and virus particles. Furthermore, the regression of SNAP25 did not affect the initial infection of the virus but reduced the infectivity of progeny virions.

Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination.

Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3′ exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2‐NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2‐NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus‐strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV‐DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

Dengue virus (DENV) causes frequent epidemics infecting ∼390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (∼25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with >97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

Potent Enhancement of HIV-1 Replication by Nef in the Absence of SERINC3 and SERINC5.

It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.

IFITM Genes, Variants, and Their Roles in the Control and Pathogenesis of Viral Infections.

Interferon-induced transmembrane proteins (IFITMs) are a family of small proteins that localize in the plasma and endolysosomal membranes. IFITMs not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. Not surprisingly, some viruses can evade the restriction of IFITMs and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. Similar to many other host defense genes that evolve under the selective pressure of microorganism infection, IFITM genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (SNPs) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection (e.g., influenza A virus). Here, we review the function and potential impact of genetic variation for IFITM restriction of viral infections. Continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among IFITMs and viruses in an effort to determine their pathobiological roles in the context of viral infections in vivo.

Host oxidative folding pathways offer novel anti-chikungunya virus drug targets with broad spectrum potential

Alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins, and likely depend on host protein disulfide isomerase-family enzymes (PDI) to aid in facilitating disulfide bond formation and isomerization in these proteins. Here, we show that in human HEK293 cells, commercially available inhibitors of PDI or modulators thereof (thioredoxin reductase, TRX-R; endoplasmic reticulum oxidoreductin-1, ERO-1) inhibit the replication of CHIKV chikungunya virus (CHIKV) in vitro in a dose-dependent manner. Further, the TRX-R inhibitor auranofin inhibited Venezuelan equine encephalitis virus and the flavivirus Zika virus replication in vitro, while PDI inhibitor 16F16 reduced replication but demonstrated notable toxicity. 16F16 significantly altered the viral genome: plaque-forming unit (PFU) ratio of CHIKV in vitro without affecting relative intracellular viral RNA quantities and inhibited CHIKV E1-induced cell-cell fusion, suggesting that PDI inhibitors alter progeny virion infectivity through altered envelope function. Auranofin also increased the extracellular genome:PFU ratio but decreased the amount of intracellular CHIKV RNA, suggesting an alternative mechanism of action. Finally, auranofin reduced footpad swelling and viremia in the C57BL/6 murine model of CHIKV infection. Our results suggest that targeting oxidative folding pathways represents a potential new anti-alphavirus therapeutic strategy.

Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCFcyclin F E3 Ligase Machinery

Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCFcyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCFcyclin F E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.

Inhibition of enterovirus VP4 myristoylation is a potential antiviral strategy for hand, foot and mouth disease

The Hand, Foot and Mouth Disease (HFMD) can result from infections by a plethora of human enteroviruses of the species Enterovirus A and B. These infections are highly contagious, resulting in regular outbreaks especially in the Asia-Pacific Region in the recent decade. Although this disease is generally a childhood affliction which manifests as a mild, febrile illness accompanied by the vesicles on the hands, feet and mouth, permanent morbidity or even fatality can result from severe forms of the disease in a subset of the infected patients. The N-terminal myristoylation signal (MGXXXS) of viral capsid protein VP4, one of the four viral structural proteins, is an extremely well conserved feature of enteroviruses, a potential antiviral target that may yield broad-spectrum inhibitors of HFMD. In this study, we have confirmed through the use of small interfering RNAs, human N-myristoyltransferase 1 plays an integral role in human Enterovirus 71 replication. Subsequent studies by inhibition of myristoylation using different myristic acid analogues elicited differential effects on the virus replication in human rhabdomyosarcoma cells. In particular, 2-hydroxymyristic acid specifically inhibited the cleavage between VP4 and VP2, part of the virion maturation process required to ensure infectivity of progeny virions while 4-oxatetradecanoic acid reduced the synthesis of viral RNA. These findings suggest that the requirement of a myristate moiety in viral structural protein precursor cleavage can serve as a viable antiviral target for further research.

The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding.

HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release.

DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity

Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5′-untranslated region (5′-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5′-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5′-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5′-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5′-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS‐segment of the HIV-1 5′-UTR during virus assembly.

The activity of Nef on HIV-1 infectivity.

The replication and pathogenicity of lentiviruses is crucially modulated by “auxiliary proteins” which are expressed in addition to the canonical retroviral ORFs gag, pol, and env. Strategies to inhibit the activity of such proteins are often sought and proposed as possible additions to increase efficacy of the traditional antiretroviral therapy. This requires the acquisition of an in-depth knowledge of the molecular mechanisms underlying their function. The Nef auxiliary protein is expressed uniquely by primate lentiviruses and plays an important role in virus replication in vivo and in the onset of AIDS. Among its several activities Nef enhances the intrinsic infectivity of progeny virions through a mechanism which remains today enigmatic. Here we review the current knowledge surrounding such activity and we discuss its possible role in HIV biology.

The Nef-Like Effect of Murine Leukemia Virus Glycosylated Gag on HIV-1 Infectivity Is Mediated by Its Cytoplasmic Domain and Depends on the AP-2 Adaptor Complex

Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

Apolipoprotein L1 (APOL1) is a major component of the human innate immune response against African trypanosomes. Although the mechanism of the trypanolytic activity of circulating APOL1 has been recently clarified, the intracellular function(s) of APOL1 in human cells remains poorly defined. Like that of many genes linked to host immunity, APOL1 expression is induced by proinflammatory cytokines gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Additionally, IFN-γ-polarized macrophages that potently restrict HIV-1 replication express APOL1, which suggests that APOL1 may contribute to HIV-1 suppression. Here, we report that APOL1 inhibits HIV-1 replication by multiple mechanisms. We found that APOL1 protein targeted HIV-1 Gag for degradation by the endolysosomal pathway. Interestingly, we found that APOL1 stimulated both endocytosis and lysosomal biogenesis by promoting nuclear localization of transcription factor EB (TFEB) and expression of TFEB target genes. Moreover, we demonstrated that APOL1 depletes cellular viral accessory protein Vif, which counteracts the host restriction factor APOBEC3G, via a pathway involving degradation of Vif in lysosomes and by secretion of Vif in microvesicles. As a result of Vif depletion by APOL1, APOBEC3G was not degraded and reduced infectivity of progeny virions. In support of this model, we also showed that endogenous expression of APOL1 in differentiated U937 monocytic cells stimulated with IFN-γ resulted in a reduced production of virus particles. This finding supports the hypothesis that induction of APOL1 contributes to HIV-1 suppression in differentiated monocytes. Deciphering the precise mechanism of APOL1-mediated HIV-1 restriction may facilitate the design of unique therapeutics to target HIV-1 replication.

UPF1 is crucial for the infectivity of human immunodeficiency virus type 1 progeny virions.

The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.

LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

BackgroundLEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN.ResultsHere we demonstrate that LEDGINs reduce the replication capacity of HIV particles produced in their presence. We systematically studied the molecular basis of this late effect of LEDGINs and demonstrate that HIV virions produced in their presence display a severe replication defect. Both the late effect and the previously described, early effect on integration contribute to LEDGIN antiviral activity as shown by time-of-addition, qPCR and infectivity assays. The late effect phenotype requires binding of LEDGINs to integrase without influencing proteolytic cleavage or production of viral particles. LEDGINs augment IN multimerization during virion assembly or in the released viral particles and severely hamper the infectivity of progeny virions. About 70% of the particles produced in LEDGIN-treated cells do not form a core or display aberrant empty cores with a mislocalized electron-dense ribonucleoprotein. The LEDGIN-treated virus displays defective reverse transcription and nuclear import steps in the target cells. The LEDGIN effect is possibly exerted at the level of the Pol precursor polyprotein.ConclusionOur results suggest that LEDGINs modulate IN multimerization in progeny virions and impair the formation of regular cores during the maturation step, resulting in a decreased infectivity of the viral particles in the target cells. LEDGINs thus profile as unique antivirals with combined early (integration) and late (IN assembly) effects on the HIV replication cycle.