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The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Prabhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag-PM interactions and CA assembly.

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription., (© 2024. The Author(s).)

Endogenous retroviruses (ERVs) are involved in autoimmune diseases such as type 1 diabetes (T1D). ERV gene products homologous to murine leukemia retroviruses are expressed in the pancreatic islets of NOD mice, a model of T1D. One ERV gene, Gag, with partial or complete open reading frames (ORFs), is detected in the islets, and it contains many sequence variants. An amplicon deep sequencing analysis was established by targeting a conserved region within the Gag gene to compare NOD with T1D-resistant mice or different ages of prediabetic NOD mice. We observed that the numbers of different Gag variants and ORFs are linked to T1D susceptibility. More importantly, these numbers change during the course of diabetes development and can be quantified to calculate the levels of disease progression. Sequence alignment analysis led to identification of additional markers, including nucleotide mismatching and amino acid consensus at specific positions that can distinguish the early and late stages, before diabetes onset. Therefore, the expression of sequence variants and ORFs of ERV genes, particularly Gag, can be quantified as biomarkers to estimate T1D susceptibility and disease progression., (Copyright © 2024 by The American Association of Immunologists, Inc.)

The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine.

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology., (© 2024. The Author(s).)

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs. These differences in Gag multimerization were further shown to affect downstream selective protein sorting into HIV membranes, indicating that the choice of RNA for packaging can modulate viral membrane compositions. These findings should advance the understanding of HIV assembly and further benefit the development of virus-like particle-based therapeutics.

Small ruminant lentiviruses (SRLVs), are grouped in Retroviridae family, remain a significant loss in the small ruminant husbandry. As a result of unavailability of vaccine and effective treatment, the diagnosis plays a crucial role for the control of SRLV infection. However, the major challenge of diagnosis of SRLV infection is the genetic and antigenic variability of the viruses that can lead to a failure in serological detection. This study investigated the circulating strains of the viruses in goats in Thailand and an in-house ELISA was developed. The coding sequences for gag protein were optimized, synthesized, and expressed in Escherichia coli for increasing the sensitivity of ELISA test. A total of 365 serum samples were examined against the recombinant protein in an in-house ELISA. The results showed that the recombinant gag achieves 96.67% sensitivity and 93.18% specificity as compared with the commercially available ELISA test kit., (© 2024. The Author(s).)

During the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-genomic RNA (gRNA) interactions play a crucial role in the multimerization process, which is yet to be fully understood. We performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model applied to the simulated trajectories, is observed to be altered by implicit gRNA binding, as well as the multimerization state of the Gag. The lateral dynamics of our simulated membrane-bound Gag proteins, with and without gRNA binding, agree with prior experimental data and help to validate our simulation models and methods. The gRNA binding is observed to affect mainly the SP1 domain of the 18-mer and the matrix-capsid linker domain of the hexamer. In the absence of gRNA binding, the independent dynamical motion of the nucleocapsid domain results in a collapsed state of the dimeric Gag. Unlike stable SP1 helices in the six-helix bundle, without IP6 binding, the SP1 domain undergoes a spontaneous helix-to-coil transition in the dimeric Gag. Together, our findings reveal conformational switches of Gag at different stages of the multimerization process and predict that the gRNA binding reinforces an efficient binding surface of Gag for multimerization, and also regulates the dynamic organization of the local membrane region itself., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Activity-regulated cytoskeleton-associated (Arc) protein is predominantly expressed in excitatory glutamatergic neurons of vertebrates, where it plays a pivotal role in regulation of synaptic plasticity. Arc protein forms capsid-like particles, which can encapsulate and transfer mRNA in extracellular vesicles (EVs) between hippocampal neurons. Once glioma cell networks actively interact with neurons via paracrine signaling and formation of neurogliomal glutamatergic synapses, we predicted the involvement of Arc in a process of EV-mediated mRNA transfer between glioma cells., Materials and Methods: Arc expression in three human glioma cell lines was evaluated by WB and immunocytochemistry. The properties of Arc protein/mRNA-containing EVs produced by glioma cells were analyzed by RT-PCR, TEM, and WB. Flow cytometry, RT-PCR, and fluorescent microscopy were used to show the involvement of Arc in EV-mediated mRNA transfer between glioma cells., Results: It was found that human glioma cells can produce EVs containing Arc/Arg3.1 protein and Arc mRNA (or "Arc EVs"). Arc EVs from U87 glioma cells internalize and deliver Arc mRNA to recipient U87 cells, where it is translated into a protein. Arc overexpression significantly increases EV production, alters EV morphology, and enhances intercellular transfer of highly expressed mRNA in glioma cell culture., Conclusion: These findings indicate involvement of Arc EVs into mRNA transfer between glioma cells that could contribute to tumor progression and affect synaptic plasticity in cancer patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region- positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development., (© 2023. The Author(s).)

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs., (© 2023. Pleiades Publishing, Ltd.)

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus.

Maedi Visna Virus (MVV) causes a chronic viral disease in sheep. Since there is no specific therapeutic drug that targets MVV, development of a vaccine against the MVV is inevitable. This study aimed to analyze the gag and env proteins as vaccine candidate proteins and to identify epitopes in these proteins. In addition, it was aimed to construct a multi-epitope vaccine candidate. According to the obtained results, the gag protein was detected to be more conserved and had a higher antigenicity value. Also, the number of alpha helix in the secondary structure was higher and transmembrane helices were not detected. Although many B cell and MHC-I/II epitopes were predicted, only 19 of them were detected to have the properties of antigenic, non-allergenic, non-toxic, soluble, and non-hemolytic. Of these epitopes, five were remarkable due to having the highest antigenicity value. However, the final multi-epitope vaccine was constructed with 19 epitopes. A strong affinity was shown between the final multi-epitope vaccine and TLR-2/4. In conclusion, the gag protein was a better antigen. However, both proteins had epitopes with high antigenicity value. Also, the final multi-epitope vaccine construct had a potential to be used as a peptide vaccine due to its immuno-informatics results., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal pr ion- l ike d omain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.

In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of Gag polyproteins induce membrane deformation and budding. Release of the virion requires direct interaction between immature Gag lattice and upstream ESCRT machinery at the viral budding site, followed by assembly of downstream ESCRT-III factors, culminating in membrane scission. However, molecular details of upstream ESCRT assembly dynamics at the viral budding site remain unclear. In this work, using coarse-grained (CG) molecular dynamics (MD) simulations, we investigated the interactions between Gag, ESCRT-I, ESCRT-II, and membrane to delineate the dynamical mechanisms by which upstream ESCRTs assemble templated by late-stage immature Gag lattice. We first systematically derived "bottom-up" CG molecular models and interactions of upstream ESCRT proteins from experimental structural data and extensive all-atom MD simulations. Using these molecular models, we performed CG MD simulations of ESCRT-I oligomerization and ESCRT-I/II supercomplex formation at the neck of the budding virion. Our simulations demonstrate that ESCRT-I can effectively oligomerize to higher-order complexes templated by the immature Gag lattice both in the absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes formed in our simulations exhibit predominantly columnar structures, which has important implications for the nucleation pathway of downstream ESCRT-III polymers. Importantly, ESCRT-I/II supercomplexes bound to Gag initiate membrane neck constriction by pulling the inner edge of the bud neck closer to the ESCRT-I headpiece ring. Our findings serve to elucidate a network of interactions between upstream ESCRT machinery, immature Gag lattice, and membrane neck that regulate protein assembly dynamics at the HIV-1 budding site., Competing Interests: Declaration of interests J.H.H. is a co-founder and shareholder of Casma Therapeutics and receives research funding from Casma Therapeutics, Genentech, and Hoffmann-La Roche., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

Telomeres in Drosophila melanogaster , which have inspired a large part of Sergio Pimpinelli work, are similar to those of other eukaryotes in terms of their function. Yet, their length maintenance relies on the transposition of the specialized retrotransposons Het-A , TART , and TAHRE , rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Our previous work has led to the isolation of a dominant mutation, Tel 1 , that caused a several-fold elongation of telomeres. In this study, we molecularly identified the Tel 1 mutation by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel 1 to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel 1 to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel 1 telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.

(1) Background: HIV-1 sub-subtype A1 is common in parts of Africa, Russia, former Soviet Union countries, and Eastern Europe. In Pakistan, sub-subtype A1 is the predominant HIV-1 subtype. Preliminary evidence suggests that distinct strains of HIV-1 sub-subtype A1 are circulating in Pakistan; however, an in-depth molecular phylogenetic characterization of HIV-1 sub-subtype A1 strains in Pakistan have not been presented. We performed a detailed characterization of the HIV-1 sub-subtype A1 epidemic in Pakistan using state-of-the-art molecular epidemiology and phylodynamics. (2) Methods: A total of 143 HIV-1 sub-subtype A1 gag sequences, including 61 sequences generated specifically for this study from PLHIVs part of our cohort, representing all sub-subtype A1 gag sequences from Pakistan, were analyzed. Maximum-likelihood phylogenetic cluster analysis was used to determine the relationship between Pakistani sub-subtype A1 strains and pandemic sub-subtype A1 strains. Furthermore, we used signature variation, charge distribution, selection pressures, and epitope prediction analyses to characterize variations unique to Pakistani HIV-1 strains and establish the association between signature variations and Gag epitope profile. (3) Results: The HIV-1 sub-subtype A1 sequences from Pakistan formed three main clusters: two that clustered with Kenyan sequences (7 and 10 sequences, respectively) and one that formed a Pakistan-specific cluster of 123 sequences that were much less related to other sub-subtype A1 sequences available in the database. The sequences in the Pakistan-specific cluster and the Kenyan reference strains exhibited several signature variations, especially at amino acid positions 312, 319, 331, 372, 373, 383, and 402. Structural protein modeling suggested that amino acid changes in these positions result in alterations of the Gag protein structure as well as in Gag-specific T-cell epitopes. (4) Conclusions: Our results suggest that the majority of the Pakistan HIV-1 sub-subtype A1 strains were unique to Pakistan and with a specific mutation pattern in Gag.

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease., (© 2022. The Author(s).)

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P 2 . We have recently shown that PI(4,5)P 2 binds directly to HTLV-1 unmyristoylated MA [myr(-)MA] and that myr(-)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P 2 . Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA-membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P 2 -binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P 2 . Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions in vivo and immature virus-like particles in vitro . To date, however, several distinct mechanistic roles for IP6 have been proposed on the basis of independent functional, structural, and kinetic studies. In this work, we investigate the molecular influence of IP6 on the structural outcomes and dynamics of CA/SP1 assembly using coarse-grained (CG) molecular dynamics (MD) simulations and free energy calculations. Here, we derive a bottom-up, low-resolution, and implicit-solvent CG model of CA/SP1 and IP6, and simulate their assembly under conditions that emulate both in vitro and in vivo systems. Our analysis identifies IP6 as an assembly accelerant that promotes curvature generation and fissure-like defects throughout the lattice. Our findings suggest that IP6 induces kinetically trapped immature morphologies, which may be physiologically important for later stages of viral morphogenesis and potentially useful for virus-like particle technologies.

Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Vaccine-induced protective T cell immunity is necessary for HIV-1 functional cure. We previously reported that rhesus PD1-Gag-based DNA vaccination sustained simian-human immunodeficiency virus (SHIV) suppression by inducing effector-memory CD8 + T cells. Here, we investigated a human PD1-Gag-based DNA vaccine, namely, ICVAX, for clinical translation. PD1-based dendritic cell targeting and mosaic antigenic designs were combined to generate the ICVAX by fusing the human soluble PD1 domain with a bivalent HIV-1 Gag-p41 mosaic antigen. The mosaic antigen was cross-reactive with patients infected with B, CRF07/08_BC, and CRF01_AE variants. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses than mosaic Gag-p41 alone, and suppressed EcoHIV infection more efficiently. In macaques, ICVAX elicited polyfunctional effector-memory T cell responses that targeted multiple nonoverlapping epitopes of the Gag-p41 antigen. Furthermore, ICVAX manufactured following good manufacturing practices proved potent immunogenicity in macaques after biannual homologous vaccination, warranting clinical evaluation of ICVAX as an immunotherapy against HIV-1. IMPORTANCE This study presents that ICVAX, a PD1-based DNA vaccine against HIV-1, could induce broad and polyfunctional T cell responses against different HIV-1 subtypes. ICVAX encodes a recombinant antigen consisting of the human soluble PD1 domain fused with two mosaic Gag-p41 antigens. The mosaic antigens cover more than 500 HIV-1 strains circulating in China including the subtypes B/B', CRF01_AE, and CRF07/08_BC. In mice, ICVAX elicited stronger, broader, and more polyfunctional T cell responses, with better EcoHIV suppression than the nontargeting mosaic Gag-p41 DNA vaccine. Moreover, both lab-generated and GMP-grade ICVAX also elicited strong polyfunctional effector-memory T cell responses in rhesus macaques with good immunogenicity against multiple nonoverlapping epitopes of the Gag-p41 antigen. This study therefore highlights the great potential to translate the PD1-based DNA vaccine approach into clinical use, and opens up new avenues for alternative HIV-1 vaccine design for HIV-1 preventive and functional cure.

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.

Nucleocapsid protein (NC) in the group-specific antigen (gag) of retrovirus is essential in the interactions of most retroviral gag proteins with RNAs. Computational method to predict NCs would benefit subsequent structure analysis and functional study on them. However, no computational method to predict the exact locations of NCs in retroviruses has been proposed yet. The wide range of length variation of NCs also increases the difficulties. In this paper, a computational method to identify NCs in retroviruses is proposed. All available retrovirus sequences with NC annotations were collected from NCBI. Models based on random forest (RF) and weighted support vector machine (WSVM) were built to predict initiation and termination sites of NCs. Factor analysis scales of generalized amino acid information along with position weight matrix were utilized to generate the feature space. Homology based gene prediction methods were also compared and integrated to bring out better predicting performance. Candidate initiation and termination sites predicted were then combined and screened according to their intervals, decision values and alignment scores. All available gag sequences without NC annotations were scanned with the model to detect putative NCs. Geometric means of sensitivity and specificity generated from prediction of initiation and termination sites under fivefold cross-validation are 0.9900 and 0.9548 respectively. 90.91% of all the collected retrovirus sequences with NC annotations could be predicted totally correct by the model combining WSVM, RF and simple alignment. The composite model performs better than the simplex ones. 235 putative NCs in unannotated gags were detected by the model. Our prediction method performs well on NC recognition and could also be expanded to solve other gene prediction problems, especially those whose training samples have large length variations., (© 2022. The Author(s).)

The Gag proteins of retroviruses play an essential role in virus particle assembly by forming a protein shell or capsid and thus generating the virion compartment. A variety of human proteins have now been identified with structural similarity to one or more of the major Gag domains. These human proteins are thought to have been evolved or "domesticated" from ancient integrations due to retroviral infections or retrotransposons. Here, we report that X-ray crystal structures of stably folded domains of MOAP1 (modulator of apoptosis 1) and PEG10 (paternally expressed gene 10) are highly similar to the C-terminal capsid (CA) domains of cognate Gag proteins. The structures confirm classification of MOAP1 and PEG10 as domesticated Gags, and suggest that these proteins may have preserved some of the key interactions that facilitated assembly of their ancestral Gags into capsids., (© 2021 Wiley Periodicals LLC.)

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

Endogenous retroviruses (ERVs) occupy a substantial fraction of mammalian genomes. However, whether ERVs extensively exist in ancient vertebrates remains unexplored. Here, we performed a genome-wide characterization of ERVs in a zebrafish (Danio rerio) model. Approximately 3,315 ERV-like elements ( Dr ERVs) were identified as Gypsy, Copia, Bel, and class I-III groups. Dr ERVs accounted for approximately 2.3% of zebrafish genome and were distributed in all 25 chromosomes, with a remarkable bias on chromosome 4. Gypsy and class I are the two most abundant groups with earlier insertion times. The vast majority of the Dr ERVs have varied structural defects. A total of 509 gag and 71 env genes with coding potentials were detected. The env -coding elements were well-characterized and classified into four subgroups. A ERV-E4.8.43-DanRer element shows high similarity with HERV9NC-int in humans and analogous sequences were detected in species spanning from fish to mammals. RNA-seq data showed that hundreds of Dr ERVs were expressed in embryos and tissues under physiological conditions, and most of them exhibited stage and tissue specificity. Additionally, 421 Dr ERVs showed strong responsiveness to virus infection. A unique group of Dr ERVs with immune-relevant genes, such as fga , ddx41, ftr35, igl1c3 , and tbk1 , instead of intrinsic viral genes were identified. These Dr ERVs are regulated by transcriptional factors binding at the long terminal repeats. This study provided a survey of the composition, phylogeny, and potential functions of ERVs in a fish model, which benefits the understanding of the evolutionary history of ERVs from fish to mammals. IMPORTANCE Endogenous retroviruses (ERVs) are relics of past infection that constitute up to 8% of the human genome. Understanding the genetic evolution of the ERV family and the interplay of ERVs and encoded RNAs and proteins with host function has become a new frontier in biology. Fish, as the most primitive vertebrate host for retroviruses, is an indispensable integral part for such investigations. In the present study, we report the genome-wide characterization of ERVs in zebrafish, an attractive model organism of ancient vertebrates from multiple perspectives, including composition, genomic organization, chromosome distribution, classification, phylogeny, insertion time, characterization of gag and env genes, and expression profiles in embryos and tissues. The result helps uncover the evolutionarily conserved and fish-specific ERVs, as well as the immune-relevant ERVs in response to virus infection. This study demonstrates the previously unrecognized abundance, diversification, and extensive activity of ERVs at the early stage of ERV evolution.

Staufen, the RNA-binding family of proteins, affects various steps in the Human Immuno-Deficiency Virus (HIV-1) replication cycle. While our previous study established Staufen-2-HIV-1 Rev interaction and its role in augmenting nucleocytoplasmic export of RRE-containing viral RNA, viral incorporation of Staufen-2 and its effect on viral propagation were unknown. Here, we report that Staufen-2 interacts with HIV-1 Gag and is incorporated into virions and that encapsidated Staufen-2 boosted viral infectivity. Further, Staufen-2 gets co-packaged into virions, possibly by interacting with host factors Staufen-1 or antiviral protein APOBEC3G, which resulted in different outcomes on the infectivity of Staufen-2-encapsidated virions. These observations suggest that encapsidated host factors influence viral population dynamics and infectivity. With the explicit identification of the incorporation of Staufen proteins into HIV-1 and other retroviruses, such as Simian Immunodeficiency Virus (SIV), we propose that packaging of RNA binding proteins, such as Staufen, in budding virions of retroviruses is probably a general phenomenon that can drive or impact the viral population dynamics, infectivity, and evolution.

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed ( P  < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

In this study, we investigated how Staufen1 influences the HIV-1 production. The overexpression of Staufen1 increased virus production without any negative affect on the viral infectivity. This increase was not caused by transcriptional activation; but by influencing post-transcriptional steps. Using multiple Gag protein derivatives, we confirmed that the zinc-finger domains of the HIV-1 nucleocapsid (NC) are important for its interaction with Staufen1. We also found that Staufen1 colocalized in stress granules with the mature form of the HIV-1 NC protein. [BMB Reports 2021; 54(11): 551-556].

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex" retroviruses, such as human immunodeficiency virus (HIV), use cis -acting elements on the unspliced gRNA in conjunction with trans -acting viral proteins to facilitate this escape. "Simple" retroviruses, such as Mason-Pfizer monkey virus (MPMV) and murine leukemia virus (MLV), exclusively use cis -acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous sarcoma virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling-deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to wild-type (WT) virus and surprisingly is replication competent, albeit with a slower rate of spread than observed in WT virus. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. IMPORTANCE While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. Rous sarcoma virus (RSV) Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis - and trans -acting mechanisms for gRNA export.

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism., (© 2021. The Author(s).)

Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag-PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P 2 than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P 2 via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P 2 and phosphatidylserine enhance myr(-)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(-)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo . The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

Background: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses., Results: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution., Conclusions: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years., (© 2021. The Author(s).)

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. IMPORTANCE Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus occurs only upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78 Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The human APOBEC3A (A3A) polynucleotide cytidine deaminase has been shown to have antiviral activity against HTLV-1 but not HIV-1, when expressed in the virus producer cell. In viral target cells, high levels of endogenous A3A activity have been associated with the restriction of HIV-1 during infection. Here we demonstrate that A3A derived from both target cells and producer cells can block the infection of Moloney-MLV (MLV) and related AKV-derived strains of MLV in a deaminase-dependent mode. Furthermore, glycosylated Gag (glycoGag) of MLV inhibits the encapsidation of human A3A, but target cell A3A was not affected by glycoGag and exerted deamination of viral DNA. Importantly, our results clearly indicate that poor glycoGag expression in MLV gag-pol packaging constructs as compared to abundant levels in full-length amphotropic MLV makes these viral vectors sensitive to A3A-mediated restriction. This raises the possibility of acquiring A3A-induced mutations in retroviral gene therapy applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Viruses and transposable elements are major drivers of evolution and make up over half the sequences in the human genome. In some cases, these elements are co-opted to perform biological functions for the host. Recent studies made the surprising observation that the neuronal gene Arc forms virus-like protein capsids that can transfer RNA between neurons to mediate a novel intercellular communication pathway. Phylogenetic analyses showed that mammalian Arc is derived from an ancient retrotransposon of the Ty3/gypsy family and contains homology to the retroviral Gag polyproteins. The Drosophila Arc homologs, which are independently derived from the same family of retrotransposons, also mediate cell-to-cell signaling of RNA at the neuromuscular junction; a striking example of convergent evolution. Here we propose an Arc 'life cycle', based on what is known about retroviral Gag, and discuss how elucidating these biological processes may lead to novel insights into brain plasticity and memory., Competing Interests: Declaration of Interests J.D.S is a scientific cofounder and consultant to VNV, with a patent application related to this work., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Patient-derived xenografts are crucial for drug development but their use is challenged by issues such as murine viral infection. We evaluate the scope of viral infection and its impact on patient-derived xenografts by taking an unbiased data-driven approach to analyze unmapped RNA-Seq reads from 184 experiments. We find and experimentally validate the extensive presence of murine viral sequence reads covering entire viral genomes in patient-derived xenografts. The existence of viral sequences inside tumor cells is further confirmed by single cell sequencing data. Extensive chimeric reads containing both viral and human sequences are also observed. Furthermore, we find significantly changed expression levels of many cancer-, immune-, and drug metabolism-related genes in samples with high virus load. Our analyses indicate a need to carefully evaluate the impact of viral infection on patient-derived xenografts for drug development. They also point to a need for attention to quality control of patient-derived xenograft experiments.

Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections., (Copyright © 2021 American Society for Microbiology.)

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer ( n = 27, median age 65.2 years (range 47-70)) and compared to prostate cancer-free male controls ( n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men ( p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.

The search for a preventive vaccine against HIV infection remains an ongoing challenge, indicating the need for novel approaches. Parainfluenza virus 5 (PIV5) is a paramyxovirus replicating in the upper airways that is not associated with any animal or human pathology. In animal models, PIV5-vectored vaccines have shown protection against influenza, RSV, and other human pathogens. Here, we generated PIV5 vaccines expressing HIV envelope (Env) and SIV Gag and administered them intranasally to macaques, followed by boosting with virus-like particles (VLPs) containing trimeric HIV Env. Moreover, we compared the immune responses generated by PIV5-SHIV prime/VLPs boost regimen in naïve vs a control group in which pre-existing immunity to the PIV5 vector was established. We demonstrate for the first time that intranasal administration of PIV5-based HIV vaccines is safe, well-tolerated and immunogenic, and that boosting with adjuvanted trimeric Env VLPs enhances humoral and cellular immune responses. The PIV5 prime/VLPs boost regimen induced robust and durable systemic and mucosal Env-specific antibody titers with functional activities including ADCC and neutralization. This regimen also induced highly polyfunctional antigen-specific T cell responses. Importantly, we show that diminished responses due to PIV5 pre-existing immunity can be overcome in part with VLP protein boosts. Overall, these results establish that PIV5-based HIV vaccine candidates are promising and warrant further investigation including moving on to primate challenge studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xiao, Dienger-Stambaugh, Chen, Wei, Phan, Beavis, Singh, Adhikary, Tiwari, Villinger, He and Spearman.)