Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

Computational method for designing vaccines applied to virus-like particles (VLPs) as epitope carriers.

Nonenveloped virus-like particles (VLPs) are self-assembled oligomeric structures composed of one or more proteins that originate from diverse viruses. Because these VLPs have similar antigenicity to the parental virus, they are successfully used as vaccines against cognate virus infection. Furthermore, after foreign antigenic sequences are inserted in their protein components (chimVLPs), some VLPs are also amenable to producing vaccines against pathogens other than the virus it originates from (these VLPs are named platform or epitope carrier). Designing chimVLP vaccines is challenging because the immunogenic response must be oriented against a given antigen without altering stimulant properties inherent to the VLP. An important step in this process is choosing the location of the sequence modifications because this must be performed without compromising the assembly and stability of the original VLP. Currently, many immunogenic data and computational tools can help guide the design of chimVLPs, thus reducing experimental costs and work. In this study, we analyze the structure of a novel VLP that originate from an insect virus and describe the putative regions of its three structural proteins amenable to insertion. For this purpose, we employed molecular dynamics (MD) simulations to assess chimVLP stability by comparing mutated and wild-type (WT) VLP protein trajectories. We applied this procedure to design a chimVLP that can serve as a prophylactic vaccine against the SARS-CoV-2 virus. The methodology described in this work is generally applicable for VLP-based vaccine development.

From North/Central America to the World? Assessing the potential of Pycnoderes quadrimaculatus Guerin-Meneville (Heteroptera: Miridae) as a pest through Ecological Niche Models.

BACKGROUND: Pycnoderes quadrimaculatus is a pest that feeds on several plants, many of which are economically important. It is native to North/Central America and its distribution has expanded to several countries in South America. RESULTS: Ecological niche models show that P. quadrimaculatus has invaded regions with climates different from those of its native range, and that there are suitable climatic conditions for its establishment worldwide. Regions where P. quadrimaculatus is a major threat and possible natural pathways of ingression were identified. In the future, its distribution will be modified by climate change. CONCLUSIONS: This study provides useful information for risk assessment and pest management of P. quadrimaculatus. According to our results, the species has great potential as a pest because it can adapt to different climatic conditions and feeds on a wide range of economically important plants. Over time, its distribution has expanded, and our models suggest that it will continue to invade other regions unless preventive measures are taken. © 2023 Society of Chemical Industry.

The 1H, 15N and 13C resonance assignments of dengue virus capsid protein with the deletion of the intrinsically disordered N-terminal region.

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Identification of 2-(4-N,N-Dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole targeting HIV-1 CA capsid protein and inhibiting HIV-1 replication in cellulo.

The capsid (CA) subunit of the HIV-1 Gag polyprotein is involved in several steps of the viral cycle, from the assembly of new viral particles to the protection of the viral genome until it enters into the nucleus of newly infected cells. As such, it represents an interesting therapeutic target to tackle HIV infection. In this study, we screened hundreds of compounds with a low cost of synthesis for their ability to interfere with Gag assembly in vitro. Representatives of the most promising families of compounds were then tested for their ability to inhibit HIV-1 replication in cellulo. From these molecules, a hit compound from the benzimidazole family with high metabolic stability and low toxicity, 2-(4-N,N-dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole (696), appeared to block HIV-1 replication with an IC50 of 3 µM. Quantitative PCR experiments demonstrated that 696 does not block HIV-1 infection before the end of reverse transcription, and molecular docking confirmed that 696 is likely to bind at the interface between two monomers of CA and interfere with capsid oligomerization. Altogether, 696 represents a promising lead molecule for the development of a new series of HIV-1 inhibitors.

The Capsid Precursor Protein of Astrovirus VA1 Is Proteolytically Processed Intracellularly.

Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.

Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus.

Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

The Use of Lectins as Tools to Combat SARS-CoV-2.

BACKGROUND: In December 2019, China announced the first case of an infection caused by an, until then, unknown virus: SARS-CoV-2. Since then, researchers have been looking for viable alternatives for the treatment and/or cure of viral infection. Among the possible complementary solutions are lectins, proteins that are reversibly bound to different carbohydrates. The Spike protein, present on the viral surface, can interact with different cell receptors: ACE2, CD147, and DC-SIGNR. Since lectins have an affinity for different carbohydrates, the binding with the glycosylated cell receptors represents a possibility of preventing the virus from binding to the receptors of host cells. OBJECTIVE: In this review, we discuss the main lectins that are possible candidates for use in the treatment of Covid-19, highlighting those that have already demonstrated antiviral activity in vivo and in vitro, including mannose-binding lectin, Griffithsin, BanLec, and others. We also aim to discuss the possible mechanism of action of lectins, which appears to occur through the mediation of viral fusion in host cells, by binding of lectins to glycosylated receptors found in human cells and/or binding of these proteins with the spike glycoprotein, present in virus surface. Moreover, we discuss the use of lectins in clinical practice. CONCLUSION: Even with the development of effective vaccines, new cases of viral infection with the same virus, or new outbreaks with different viruses can occur; so, the development of new treatments should not be discarded. Moreover, the discussions made in this work are relevant regarding the anti-viral properties of lectins.

Detection of Antibodies against Hepatitis A Virus (HAV) by a Surface Plasmon Resonance (SPR) Biosensor: A New Diagnosis Tool Based on the Major HAV Capsid Protein VP1 (SPR-HAVP1).

Hepatitis A (HA) is an acute human infectious disease caused by a positive single-stranded RNA virus (HAV). It is mainly acquired through the fecal-oral route and is primarily spread by contact between people and exposure to contaminated water and food. Recently, large outbreaks of HA have been reported by low and moderate endemicity countries, emphasizing its importance in public health and the need for rapid and large-scale diagnostic tests to support public health decisions on HA. This work proposes a new tool for HAV diagnosis based on the association of surface plasmonic resonance with major capsid protein VP1 (SPR-HAVP1 assay), detecting IgM antibodies for HAV in human serum samples. Structural analyses of VP1 B-lymphocyte epitopes showed continuous and discontinuous epitopes. The discontinuous epitopes were identified in the N-terminal region of the VP1 protein. Both epitope types in the VP1 protein were shown by the reactivity of VP1 in native and denaturing conditions to IgM anti-HAV, which was favorable to tests of VP1 in the SPR assays. SPR-HAVP1 assays showed good performance in the detection of IgM polyclonal antibody anti-HAV. These assays were performed using a COOH5 sensor chip functionalized with VP1 protein. The sensorgram record showed a significant difference between positive and negative serum samples, which was confirmed by analysis of variation of initial and final dissociation values through time (ΔRUd/t). The data gathered here are unequivocal evidence that the SPR-HAVP1 strategy can be applied to detect IgM antibodies in human serum positive to the HAV. This is a new tool to be explored to diagnose human HAV infections.

Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins.

Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed.

Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses.

Although replication-defective human adenovirus type 5 (Ad5) vectors that express in situ the capsid-encoding region of foot-and-mouth disease virus (FMDV) have been proven to be effective as vaccines in relevant species for several viral strains, the same result was not consistently achieved for the O1/Campos/Brazil/58 strain. In the present study, an optimization of the Ad5 system was explored and was proven to enhance the expression of FMDV capsid proteins and their association into virus-like particles (VLPs). Particularly, we engineered a novel Ad5 vector (Ad5[PVP2]OP) which harbors the foreign transcription unit in a leftward orientation relative to the Ad5 genome, and drives the expression of the FMDV sequences from an optimized cytomegalovirus (CMV) enhancer-promoter as well. The Ad5[PVP2]OP vaccine candidate also contains the amino acid substitutions S93F/Y98F in the VP2 protein coding sequence, predicted to stabilize FMD virus particles. Cells infected with the optimized vector showed an ∼14-fold increase in protein expression as compared to cells infected with an unmodified Ad5 vector tested in previous works. Furthermore, amino acid substitutions in VP2 protein allowed the assembly of FMDV O1/Campos/Brazil/58 VLPs. Evaluation of several serological parameters in inoculated mice with the optimized Ad5[PVP2]OP candidate revealed an enhanced vaccine performance, characterized by significant higher titers of neutralizing antibodies, as compared to our previous unmodified Ad5 vector. Moreover, 94% of the mice vaccinated with the Ad5[PVP2]OP candidate were protected from homologous challenge. These results indicate that both the optimized protein expression and the stabilization of the in situ generated VLPs improved the performance of Ad5-vectored vaccines against the FMDV O1/Campos/Brazil/58 strain and open optimistic expectations to be tested in target animals.

Antiphage activity of natural and synthetic substances: a new age for antivirals?

Viruses are considered biological entities that possess a genome and can adapt to the environment of living organisms. Since they are obligate intracellular parasites, their cycle of replication can result in cell death, and consequently, some viruses are harmful to mammalian cells and can cause disease in humans. Therefore, the search for substances for the treatment of viral diseases can be accomplished through the use of bacteriophages as models for eukaryotic cell viruses. Thus, this review highlights the main studies identifying substances with antiphage activity in comparison assays involving phages and eukaryotic viruses, in order to explore the potential of these substances as antivirals. As a future perspective, this approach may help at the beginning of an Antiviral Age.

Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organisms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible function of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates.

Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides' ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins' conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides' structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation's immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides' in vitro protection-inducing ability.

Short communication: a modified Vaccinia virus Ankara-based Porcine circovirus 2 vaccine elicits strong antibody response upon prime-boost homologous immunization in a preclinical model.

Porcine circovirus 2 (PCV2) infections are related to a number of syndromes and clinical manifestations, generally known as Porcine circovirus-associated diseases, which are related to losses in the swine industry. There are commercially available vaccines and new vaccines being tested, however, persistency of the PCV2 as an important pig pathogen, and the growing number of affected farms in different countries have suggested that there is room for vaccine improvement. In this study, we describe the construction and testing of a recombinant live vaccine based on a modified Vaccinia virus Ankara (MVA) vector expressing the PCV2b capsid protein (CAP). Using a two-dose homologous vaccination regimen, in mice, we demonstrated that the vaccine induced high titers of anti-PCV2 antibodies. The vaccine is stable upon lyophilization, and, together with the good immunogenicity potential observed, the results support further evaluation of the MVA-CAP vaccine in the target species.

Quantitation of norovirus-specific IgG before and after infection in immunocompromised patients.

Noroviruses (NoV) cause the majority of non-bacterial gastroenteritis cases worldwide, with genotype II.4 being the most common. The aim of our study was to quantitate norovirus-specific IgG in immunocompromised patients before and after laboratory-confirmed norovirus infection. A quantitative ELISA was developed by coating ELISA plates with recombinantly expressed P domain of GII.1 capsid protein. After testing mouse sera drawn before and after immunization with GII.1- and GII.4 P domain, sera from GII.1- and GII.4 infected patients were tested. The assay reliably detected preexisting NoV-specific IgG antibodies. Sera drawn after infection showed increased antibody concentrations. Antibodies elicited by GII.1- and GII.4 infections could be detected with coated GII.1 capsid protein. IgG levels remained constant during the first week and then increased in the second week after laboratory diagnosis. The results show that immunocompromised patients elicited IgG responses to NoV infections that could be reliably detected with our quantitative ELISA.

Caracterización de la proteína no capsidal 3D, del virus de la fiebre aftosa y producción de anticuerpos policlonales / Characterization of the non-capsided 3d protein, of the foot-and-mouth disease virus and the production of polyclonal antibodies

Las proteínas no capsidales del virus de la fiebre aftosa se utilizan como marcadoras en la evaluación de animales que han estado en contacto con el virus, a diferencia de los inmunizados, ya que la vacuna no debe tener estas proteínas, por lo tanto los animales no deben presentar anticuerpos contra ellas. El objetivo de esta investigación fue la caracterización de la proteína no capsidal 3D y la producción de anticuerpos policlonales in vivo. La proteína se purificó del cultivo de virus inactivo, por cromatografía de intercambio iónico. La elución de los picos fue sometida a electroforesis uni-bidimensional; demostrándose un alto grado de pureza (>90%) en el pico tres, donde se identifico la proteína 3D, por la técnica de MALDI-TOF y electroespray de trampa iónica. La proteína purificada, se inoculó en cabras y el suero hiperinmune fue precipitado y sometido a cromatografía de afinidad para la obtención de inmunoglobulinas; la reacción inmunitaria se confirmó por medio de inmunodifusión y Western blot. El proceso de purificación demostró ser eficiente y útil para la obtención de anticuerpos específicos, los cuales tendrán utilidad en la elaboración de un ensayo inmunoenzimático que mida la pureza de la vacuna frente al contenido de estas proteínas.

The noncapsid proteins of the foot and mouth disease are used as markers in the evaluation of animals that have been in contact with the virus, to discriminated the immunized animals, because the vaccine should not have these proteins, therefore animals should not present antibodies against them. The aim of this investigation was the characterization of the 3D non-capsid protein and the production of polyclonal antibodies in vivo. The protein was purified from the culture of inactivated virus, by ion exchange chromatography. The elution of the peaks were submit an one-two-dimensional electrophoresis; Demonstrated a high degree of purity (> 90%) in peak three, where the 3D protein was identified, by the MALDI-TOF technique and ion trap electrospray. The purified protein, inoculated in goats and the hyperimmune serum, was precipitate out and submitted to affinity chromatography to obtain immunoglobulins; the immune reaction was confirmed by means of immunodiffusion and Western blot. The purification process proved to be efficient and useful for obtaining specific antibodies, which will be useful in the preparation of an immunoenzymatic assay that measures the purity of the vaccine against to the content of these proteins.

The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly.

The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag-Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.