Search Results (29)

HIV-1 has evolved a plethora of strategies to overcome the cytoskeletal barrier (i.e., actin and intermediate filaments (AFs and IFs) and microtubules (MTs)) to achieve the viral cycle. HIV-1 modifies cytoskeletal organization and dynamics by acting on associated adaptors and molecular motors to productively fuse, enter, and infect cells and then traffic to the cell surface, where virions assemble and are released to spread infection. The HIV-1 envelope (Env) initiates the cycle by binding to and signaling through its main cell surface receptors (CD4/CCR5/CXCR4) to shape the cytoskeleton for fusion pore formation, which permits viral core entry. Then, the HIV-1 capsid is transported to the nucleus associated with cytoskeleton tracks under the control of specific adaptors/molecular motors, as well as HIV-1 accessory proteins. Furthermore, HIV-1 drives the late stages of the viral cycle by regulating cytoskeleton dynamics to assure viral Pr55^Gag expression and transport to the cell surface, where it assembles and buds to mature infectious virions. In this review, we therefore analyze how HIV-1 generates a cell-permissive state to infection by regulating the cytoskeleton and associated factors. Likewise, we discuss the relevance of this knowledge to understand HIV-1 infection and pathogenesis in patients and to develop therapeutic strategies to battle HIV-1. Full article

Hereditary cataracts are characterized by significant clinical and genetic heterogeneity, which can pose challenges for early DNA diagnosis. To comprehensively address this problem, it is essential to investigate the epidemiology of the disease, perform population studies to determine the spectrum and frequencies of mutations in the responsible genes, and examine clinical and genetic correlations. Based on modern concepts, non-syndromic hereditary cataracts are predominantly caused by genetic disease forms associated with mutations in crystallin and connexin genes. Therefore, a comprehensive approach to studying hereditary cataracts is necessary for early diagnosis and improved treatment outcomes. The crystallin (CRYAA, CRYAB, CRYGC, CRYGD, and CRYBA1) and connexin (GJA8, GJA3) genes were analyzed in 45 unrelated families from the Volga–Ural Region (VUR) with hereditary congenital cataracts. Pathogenic and probably pathogenic nucleotide variants were identified in ten unrelated families, nine of which had cataracts in an autosomal dominant pattern of inheritance. Two previously undescribed likely pathogenic missense variants were identified in the CRYAA gene: c.253C > T (p.L85F) in one family and c.291C > G (p.H97Q) in two families. The known mutation c.272_274delGAG (p.G91del) was found in the CRYBA1 gene in one family, while no pathogenic variants were found in the CRYAB, CRYGC, or CRYGD genes in the examined patients. In the GJA8 gene, the known mutation c.68G > C (p.R23T) was found in two families, and previously undescribed variants were identified in two other families: a c.133_142del deletion (p.W45Sfs*72) and a missense variant, c.179G > A (p.G60D). In one patient with a recessive form of cataract, two compound-heterozygous variants were identified—a previously undescribed likely pathogenic missense variant, c.143A > G (p.E48G), and a known variant with uncertain pathogenetic significance, c.741T > G (p.I24M). Additionally, a previously undescribed deletion, c.del1126_1139 (p.D376Qfs*69), was identified in the GJA3 gene in one family. In all families where mutations were identified, cataracts were diagnosed either immediately after birth or during the first year of life. The clinical presentation of the cataracts varied depending on the type of lens opacity, resulting in various clinical forms. This information emphasizes the importance of early diagnosis and genetic testing for hereditary congenital cataracts to guide appropriate management and improve outcomes. Full article

The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55^Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55^Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55^Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55^Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55^Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness. Full article

Human immunodeficiency virus 1 (HIV-1) viral protease (PR) is one of the most studied viral enzymes and a crucial antiviral target. Despite its well-characterized role in virion maturation, an increasing body of research is starting to focus on its ability to cleave host cell proteins. Such findings are apparently in contrast with the dogma of HIV-1 PR activity being restricted to the interior of nascent virions and suggest catalytic activity within the host cell environment. Given the limited amount of PR present in the virion at the time of infection, such events mainly occur during late viral gene expression, mediated by newly synthesized Gag-Pol polyprotein precursors, rather than before proviral integration. HIV-1 PR mainly targets proteins involved in three different processes: those involved in translation, those controlling cell survival, and restriction factors responsible for innate/intrinsic antiviral responses. Indeed, by cleaving host cell translation initiation factors, HIV-1 PR can impair cap-dependent translation, thus promoting IRES-mediated translation of late viral transcripts and viral production. By targeting several apoptotic factors, it modulates cell survival, thus promoting immune evasion and viral dissemination. Additionally, HIV-1 PR counteracts restriction factors incorporated in the virion that would otherwise interfere with nascent virus vitality. Thus, HIV-1 PR appears to modulate host cell function at different times and locations during its life cycle, thereby ensuring efficient viral persistency and propagation. However, we are far from having a complete picture of PR-mediated host cell modulation, which is emerging as a field that needs further investigation. Full article

HIV-1 protease (PR) is a viral enzyme that cleaves the Gag and Gag-Pol polyprotein precursors to convert them into their functional forms, a process which is essential to generate infectious viral particles. Due to its broad substrate specificity, HIV-1 PR can also cleave certain host cell proteins. Several studies have identified host cell substrates of HIV-1 PR and described the potential impact of their cleavage on HIV-1-infected cells. Of particular interest is the interaction between PR and the caspase recruitment domain-containing protein 8 (CARD8) inflammasome. A recent study demonstrated that CARD8 can sense HIV-1 PR activity and induce cell death. While PR typically has low levels of intracellular activity prior to viral budding, premature PR activation can be achieved using certain non-nucleoside reverse transcriptase inhibitors (NNRTIs), resulting in CARD8 cleavage and downstream pyroptosis. Used together with latency reversal agents, the induction of premature PR activation to trigger CARD8-mediated cell killing may help eliminate latent reservoirs in people living with HIV. This represents a novel strategy of utilizing PR as an antiviral target through premature activation rather than inhibition. In this review, we discuss the viral and host substrates of HIV-1 protease and highlight potential applications and advantages of targeting CARD8 sensing of HIV-1 PR. Full article

An increasing number of patients in Africa are experiencing virological failure on a second-line antiretroviral protease inhibitor (PI)-containing regimen, even without resistance-associated mutations in the protease region, suggesting a potential role of other genes in PI resistance. Here, we investigated the prevalence of mutations associated with Lopinavir/Ritonavir (LPV/r) failure in the Envelope gene and the possible coevolution with mutations within the Gag-protease (gag-PR) region. Env and Gag-PR sequences generated from 24 HIV-1 subtype C infected patients failing an LPV/r inclusive treatment regimen and 344 subtype C drug-naïve isolates downloaded from the Los Alamos Database were analyzed. Fisher’s exact test was used to determine the differences in mutation frequency. Bayesian network probability was applied to determine the relationship between mutations occurring within the env and gag-PR regions and LPV/r treatment. Thirty-five mutations in the env region had significantly higher frequencies in LPV/r-treated patients. A combination of Env and Gag-PR mutations was associated with a potential pathway to LPV/r resistance. While Env mutations were not directly associated with LPV/r resistance, they may exert pressure through the Gag and minor PR mutation pathways. Further investigations using site-directed mutagenesis are needed to determine the impact of Env mutations alone and in combination with Gag-PR mutations on viral fitness and LPV/r efficacy. Full article

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules. Full article

HIV-1 protease active site inhibitors are a key part of antiretroviral therapy, though resistance can evolve rendering therapy ineffective. Protease inhibitor resistance typically starts with primary mutations around the active site, which reduces inhibitor binding, protease affinity for substrate cleavage site residues P4-P4′, and viral replication. This is often followed by secondary mutations in the protease substrate-grooves which restore viral replication by increasing protease affinity for cleavage site residues P12-P5/P5′-P12′, while maintaining resistance. However, mutations in Gag alone can also result in resistance. The Gag resistance mutations can occur in cleavage sites (P12-P12′) to increase PR binding, as well as at non-cleavage sites. Here we show in silico that Gag non-cleavage site protease inhibitor resistance mutations can stabilize protease binding to Gag cleavage sites which contain structured subdomains on both sides: SP1/NC, SP2/p6, and MA/CA. The Gag non-cleavage site resistance mutations coordinated a network of H-bond interactions between the adjacent structured subdomains of the Gag substrates to form a substrate-clamp around the protease bound to cleavage site residues P12-P12′. The substrate-clamp likely slows protease disassociation from the substrate, restoring the cleavage rate in the presence of the inhibitor. Native Gag substrates can also form somewhat weaker substrate-clamps. This explains the 350-fold slower cleavage rate for the Gag CA/SP1 cleavage site in that the CA-SP1 substrate lacks structured subdomains on both sides of the cleavage site, and so cannot form a substrate-clamp around the PR. Full article

HIV-1 progeny are released from infected cells as immature particles that are unable to infect new cells. Gag-Pol polyprotein dimerization via the reverse transcriptase connection domain (RTCDs) is pivotal for proper activation of the virus protease (PR protein) in an early event of the progeny virus maturation process. Thus, the RTCD is a potential therapeutic target for a broadly effective anti-HIV agent through impediment of virus maturation. In this study, human single-chain antibodies (HuscFvs) that bound to HIV-1 RTCD were generated using phage display technology. Computerized simulation guided the selection of the transformed Escherichia coli-derived HuscFvs that bound to the RTCD dimer interface. The selected HuscFvs were linked molecularly to human-derived-cell-penetrating peptide (CPP) to make them cell-penetrable (i.e., become transbodies). The CPP-HuscFvs/transbodies produced by a selected transformed E. coli clone were tested for anti-HIV-1 activity. CPP-HuscFvs of transformed E. coli clone 11 (CPP-HuscFv11) that presumptively bound at the RTCD dimer interface effectively reduced reverse transcriptase activity in the newly released virus progeny. Infectiousness of the progeny viruses obtained from CPP-HuscFv11-treated cells were reduced by a similar magnitude to those obtained from protease/reverse transcriptase inhibitor-treated cells, indicating anti-HIV-1 activity of the transbodies. The CPP-HuscFv11/transbodies to HIV-1 RTCD could be an alternative, anti-retroviral agent for long-term HIV-1 treatment. Full article

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins. Full article

Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55^Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55^Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55^Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle. Full article

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV₃₉₂₉), including the hinge region (E273–277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV₃₉₂₉ binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV₃₉₂₉ exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV₃₉₂₉ as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV₃₉₂₉ in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273–277 and E154–156 of ALFV₃₉₂₉ were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV₃₉₂₉, with the E327/E273–277 combination most dominant. These data demonstrate that attenuation of ALFV₃₉₂₉ is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines. Full article

Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity. Full article

The human immunodeficiency virus (HIV-1) polyprotein Gag (Group-specific antigen) plays a central role in controlling the late phase of the viral lifecycle. Considered to be only a scaffolding protein for a long time, the structural protein Gag plays determinate and specific roles in HIV-1 replication. Indeed, via its different domains, Gag orchestrates the specific encapsidation of the genomic RNA, drives the formation of the viral particle by its auto-assembly (multimerization), binds multiple viral proteins, and interacts with a large number of cellular proteins that are needed for its functions from its translation location to the plasma membrane, where newly formed virions are released. Here, we review the interactions between HIV-1 Gag and 66 cellular proteins. Notably, we describe the techniques used to evidence these interactions, the different domains of Gag involved, and the implications of these interactions in the HIV-1 replication cycle. In the final part, we focus on the interactions involving the highly conserved nucleocapsid (NC) domain of Gag and detail the functions of the NC interactants along the viral lifecycle. Full article

Paternally expressed gene 10 (PEG10) is a human retrotransposon-derived imprinted gene. Previous works have demonstrated that a mutation in the coding sequence of this gene is lethal with regard to embryological age due to defects of placental development. In addition, PEG10 is implicated in several malignancies, such as pancreatic cancer and hepatocellular carcinoma. The PEG10 gene encodes two protein isoforms, which are translated by a typical retroviral frameshift mechanism. The Gag-like protein (RF1_PEG10) is encoded by reading frame 1, whilst reading frames 1 and 2 accounts for the Gag-Pol-like polyprotein (RF1/RF2_PEG10). The protease (PR) domain of RF2_PEG10 contains an -Asp-Ser-Gly- sequence, which refers to the conservative -Asp-Ser/Thr-Gly- active-site motif of retroviral aspartic proteases. The function of the aspartic protease domain of RF2_PEG10 remains unclear. In order to further investigate the function of the PEG10 protease (PR_PEG10), a frameshift mutant was generated (_fsRF1/RF2_PEG10) for comparison with the RF1/RF2_PEG10 form. To study the effects of PR_PEG10 on cellular proliferation and viability, mammalian HEK293T and HaCaT cells were transfected with plasmids encoding for either the frameshift mutant (_fsRF1/RF2_PEG10) or a PR active-site (D370A) mutant _fsRF1/RF2_PEG10. Based on our findings, an _fsRF1/RF2_PEG10 overexpression resulted in an increased cellular proliferation, compared to the mutant form. Interestingly, transfection with _fsRF1/RF2_PEG10 had a detrimental effect on cell viability. We hypothesize that PR_PEG10 may play a cardinal role in the function of this retroviral remnant, possibly implicated in cellular proliferation and the inhibition of apoptosis. Full article