A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Development of in-house ELISA based on recombinant gag proteins of small ruminant lentiviruses isolated from goats in Thailand.

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.

Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

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.

HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Conformational transitions of the HIV-1 Gag polyprotein upon multimerization and gRNA binding.

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.

Retrovirus-like gag protein Arc/Arg3.1 is involved in extracellular-vesicle-mediated mRNA transfer between glioma cells.

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.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus.

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.

HIV-1 diverts cortical actin for particle assembly and release.

Enveloped viruses assemble and bud from the host cell membranes. Any role of cortical actin in these processes have often been a source of debate. Here, we assessed if cortical actin was involved in HIV-1 assembly in infected CD4 T lymphocytes. Our results show that preventing actin branching not only increases HIV-1 particle release but also the number of individual HIV-1 Gag assembly clusters at the T cell plasma membrane. Indeed, in infected T lymphocytes and in in vitro quantitative model systems, we show that HIV-1 Gag protein prefers areas deficient in F-actin for assembling. Finally, we found that the host factor Arpin, an inhibitor of Arp2/3 branched actin, is recruited at the membrane of infected T cells and it can associate with the viral Gag protein. Altogether, our data show that, for virus assembly and particle release, HIV-1 prefers low density of cortical actin and may favor local actin debranching by subverting Arpin.

Interaction of TSG101 with the PTAP Motif in Distinct Locations of Gag Determines the Incorporation of HTLV-1 Env into the Retroviral Virion.

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.

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains.

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

In silico discovery of epitopes of gag and env proteins for the development of a multi-epitope vaccine candidate against Maedi Visna Virus using reverse vaccinology approach.

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.

An interchangeable prion-like domain is required for Ty1 retrotransposition.

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 prion-like domain (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.

Structure of the HIV immature lattice allows for essential lattice remodeling within budded virions.

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<∆G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

Dynamics of upstream ESCRT organization at the HIV-1 budding site.

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.

TDP-43 Controls HIV-1 Viral Production and Virus Infectiveness.

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 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag 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 Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag 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.

The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.

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.

Identification of the Telomere elongation Mutation in Drosophila.

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, Tel1, that caused a several-fold elongation of telomeres. In this study, we molecularly identified the Tel1 mutation by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel1 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 Tel1 to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel1 telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.

Phylogenetic Characterization of HIV-1 Sub-Subtype A1 in Karachi, Pakistan.

(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.

Occurrence of Equine Foamy Virus Infection in Horses from Poland.

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.