Tegument Protein Research Articles

Cryptic phosphoribosylase activity of NAMPT restricts the virion incorporation of viral proteins.

As obligate intracellular pathogens, viruses activate host metabolic enzymes to supply intermediates that support progeny production. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of salvage nicotinamide adenine dinucleotide (NAD+) synthesis, is an interferon-inducible protein that inhibits the replication of several RNA and DNA viruses through unknown mechanisms. Here, we show that NAMPT restricts herpes simplex virus type 1 (HSV-1) replication by impeding the virion incorporation of viral proteins owing to its phosphoribosyl-hydrolase (phosphoribosylase) activity, which is independent of the role of NAMPT in NAD+ synthesis. Proteomics analysis of HSV-1-infected cells identifies phosphoribosylated viral structural proteins, particularly glycoproteins and tegument proteins, which are de-phosphoribosylated by NAMPT in vitro and in cells. Chimeric and recombinant HSV-1 carrying phosphoribosylation-resistant mutations show that phosphoribosylation promotes the incorporation of structural proteins into HSV-1 virions and subsequent virus entry. Loss of NAMPT renders mice highly susceptible to HSV-1 infection. Our work describes an additional enzymatic activity of a metabolic enzyme in viral infection and host defence, offering a system to interrogate the roles of protein phosphoribosylation in metazoans.

TMIC-45. CYTOMEGALOVIRUS PP65 ANTIGEN EXPRESSION FOLLOWING PP65-SPECIFIC DENDRITIC CELL VACCINATION IN MATCHED PRIMARY AND RECURRENT GLIOBLASTOMA SPECIMENS

Abstract BACKGROUND Human cytomegalovirus (HCMV) expression in glioblastoma (GBM) has been corroborated by multiple independent laboratories. HCMV genes have been directly linked to GBM cell proliferation, invasion, and angiogenesis. The HCMV tegument protein pp65 has served as a tumor-specific antigen in serial pp65 RNA-pulsed autologous dendritic cell (DC) vaccine trials. Here we report pp65 expression in matched primary and recurrent GBM samples from those trials following pp65-DC vaccination. METHODS Paired GBM specimens were collected from patients receiving pp65-DC vaccines according to ATTAC and ATTAC-GM clinical trials (NCT00639639). Of 23 treated patients, 9 cases were available for analysis. Formalin-fixed paraffin-embedded specimens were mounted on glass slides and stained for pp65 via immunohistochemistry (IHC). CD31 served as a control for tissue quality and to investigate the role of pp65 co-staining in the tumor perivascular niche. RESULTS Of 9 available cases, 5 were excluded due to poor tissue quality/absent CD31 staining on aged slides. Among the remaining four cases, three showed pp65 staining in non-vascular and perivascular areas within both primary and recurrent samples. However, recurrent specimens of these positive cases demonstrated a clear reduction in both intensity and distribution of pp65 staining within non-vascular regions but retained a strong positivity within the perivascular space. The fourth case showed absent pp65 staining and only weakly positive CD31 staining in both primary and recurrent specimens. Necrotic areas within pp65-positive slides showed minimal to no pp65 staining. CONCLUSIONS This study confirms HCMV pp65 expression in both primary and recurrent GBM following pp65-DC vaccination, with an interesting phenomenon of retained pp65 expression specifically in the perivascular niche. These findings support the role of HCMV in GBM angiogenesis and tumor progression as well as mechanisms of immune evasion following single antigen vaccine platforms. Our study reinforces the importance of tissue quality for proper HCMV detection in GBM.

The loss of both pUL16 and pUL21 in HSV-1 infected cells abolishes cytoplasmic envelopment.

Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1). To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation associated recombination. The eleven fragments or "parts" spanned the 152 kb genome and recombination was achieved because of the overlapping homologous sequences between each fragment. To demonstrate the robustness of this genome assembly method for reverse genetics, we engineered different mutations that were located in distant loci on the genome and built a collection of HSV-1 genomes that contained single and different combination of mutations in 5 conserved HSV-1 genes. The five genes: UL7, UL11, UL16, UL21 and UL51 encode virion structural proteins and have varied functions in the infected cell. Each is dispensable for virus replication in cell culture, however, combinatorial analysis of deletions in the five genes revealed "synthetic-lethality" of some of the genetic mutations. Thus, it was discovered that any virus that carried a UL21 mutation in addition to the other gene was unable to replicate in Vero cells. Replication was restored in a complementing cell line that provided pUL21 in trans. One particular combination (UL16-UL21) was of interest because the proteins encoded by these genes are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1 infected cells are unclear. Both are dispensable for HSV-1 replication, however, in HSV-2 their mutation results in nuclear retention of assembled capsids. We thus characterized these viruses that carry the single and double mutant. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident but did not mature into enveloped particles. The capsid particles isolated from these cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the wild-type capsids. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16; the complex of pUL16 and pUL21 should be considered as important mediators of cytoplasmic maturation of the particle.

Suppression of ZBP1-mediated NLRP3 inflammasome by the tegument protein VP22 facilitates pseudorabies virus infection.

Z-DNA binding protein 1 (ZBP1) functions as a pivotal innate immune sensor that regulates inflammatory cell death during viral infections. However, its role in pseudorabies virus (PRV) infection remains unknown. Here, we demonstrate that ZBP1 serves as a restrictive factor by triggering the activation of the NLR family pyrin domain-containing 3 inflammasome, a process counteracted by PRV-encoded protein VP22. Furthermore, VP22 interferes with the interaction between ZBP1 and receptor-interacting protein kinase 3/Caspase-8, particularly through its N-terminal 1-50 amino acids. Importantly, deficiency in ZBP1 enhances the replication and virulence of recombinant viruses lacking VP22 or its N-terminal 1-50 amino acids. These findings reveal how PRV escapes ZBP1-mediated inflammatory responses during infection, potentially informing the rational design of therapeutic interventions.

Designing a novel multi-epitope antigen for diagnosing human cytomegalovirus infection: An immunoinformatics approach.

Human cytomegalovirus (HCMV) infection can lead to congenital infections and severe complications, particularly in immunocompromised individuals. Current serological tests for diagnosing HCMV infection often face limitations in sensitivity and specificity. Developing multi-epitope antigens for serological assays offers the potential for enhancing diagnostic accuracy. This study aimed to design a novel multi-epitope antigen for HCMV infection diagnosis using immunoinformatic approaches. Five tegument proteins (universal protein resource [UniProt] ID: Po8318, Po6725, F5HC97, Q6RX10, and F5HC05) were selected based on their antigenic properties and literature review. Six linear B-cell epitopes were predicted within conserved regions of each antigen sequence and linked with appropriate linkers. The designed multi-epitope antigen underwent thorough evaluation for physicochemical properties, solubility, antigenicity, and cross-reactivity. Additionally, the three-dimensional structure of the antigen was predicted, refined, and validated. The nucleotide sequence of the designed antigen was optimized for successful expression in Escherichia coli and inserted into a pET23a (+) vector. Immunoinformatic analysis revealed that the multi-epitope antigen exhibits stability, antigenicity, and lacks cross-reactivity. Our findings suggest that this multi-epitope antigen is a promising candidate for diagnosing HCMV infection. However, further validation through laboratory testing is required to confirm its diagnostic efficacy.

Optimisation of induction and purification protocols of recombinant 22.6kDa tegumental protein of Schistosoma spindale inprokaryotic vector

Schistosomosis is a prevalent zoonotic parasitic disease affecting both humans and animals on a global scale, with an estimated 165 million cattle and 200 million people impacted worldwide. Eventhough, serological methodologies designed for the identification of specific antibodies targeting parasitic antigens are esteemed for their high sensitivity, there are criticisms due to their incapacity to reliably indicate active infection, inability to correlate with the intensity of infection and lack of specificity. Enhancing the specificity of serological assays presents a significant challenge, primarily attributable to the identification and synthesis of specific antigens. In addressing these limitations, recombinant technology with specific immunogenic proteins as candidate antigens emerges as a viable alternative. In this study, induction of 22.6 kDa recombinant tegument protein of Schistosoma spindale was achieved using 0.6 mM concentration of IPTG at 37°C for four hours. Nickel chelating affinity chromatography was employed for protein purification, yielding maximum protein concentration at 75mM elution. Subsequently, the dialysis technique was employed to remove contaminants, while lyophilisation method was employed for protein concentration. The protein concentration post dialysis was measured at 0.220 mg/mL, while lyophilisation resulted in a concentration of 2mg/m Keywords: 22.6 kDa tegumental protein, Schistosoma spindale, dialysis, lyophilisation

Spatial transcriptomics of a parasitic flatworm provides a molecular map of drug targets and drug resistance genes

The spatial organization of gene expression dictates tissue functions in multicellular parasites. Here, we present the spatial transcriptome of a parasitic flatworm, the common liver fluke Fasciola hepatica. We identify gene expression profiles and marker genes for eight distinct tissues and validate the latter by in situ hybridization. To demonstrate the power of our spatial atlas, we focus on genes with substantial medical importance, including vaccine candidates (Ly6 proteins) and drug resistance genes (glutathione S-transferases, ABC transporters). Several of these genes exhibit unique expression patterns, indicating tissue-specific biological functions. Notably, the prioritization of tegumental protein kinases identifies a PKCβ, for which small-molecule targeting causes parasite death. Our comprehensive gene expression map provides unprecedented molecular insights into the organ systems of this complex parasitic organism, serving as a valuable tool for both basic and applied research.

Functional genomic analysis of the 68-1 RhCMV-Mycobacteria tuberculosis vaccine reveals an IL-15 response signature that is conserved with vector attenuation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection.

Duck plague virus UL24 protein initiates K48/K63-linked IRF7 polyubiquitination to antagonize the innate immune response

Duck plague virus (DPV), which is the causative agent of duck viral enteritis, is highly infectious and can cause severe disease and death in ducks, geese and other waterfowl. Several tegument proteins of DPV have been shown to affect the cyclic GMP-AMP synthase (cGAS)-STING signaling pathway to modulate host innate immune responses. DPV UL24, an important DPV tegument protein, can inhibit the activity of the IFN-β promoter. However, the mechanism by which the DPV UL24 protein regulates the host innate immune response remains unclear. In this study, we found that the UL24 protein can significantly inhibit the activity of the interferon-β promoter induced by poly(I:C) and reduce the production of IFN-β, interferon-stimulated genes (OASL, Mx), and the cellular inflammatory factor IL-6. (2) The UL24 protein can widely inhibit the mRNA level of immune signaling molecules. The UL24 protein can also downregulate the protein expression of RIG-I, MDA5, MAVS, cGAS, STING, TBK1 and IRF7 in DEFs. RT-qPCR results revealed that UL24 significantly inhibited the mRNA accumulation for the immune signaling molecules cGAS, STING, TBK1 and IRF7. (3) The UL24 protein induced the degradation of IRF7 via ubiquitination. After the DEFs were treated with the ubiquitin proteasome inhibitor MG132, the degradation of IRF7 by the UL24 protein was alleviated. Coimmunoprecipitation results revealed that DPV UL24 induced the K48/K63-linked ubiquitination of IRF7, which promoted its degradation and thus antagonized the host innate immune response.

Disruption of herpes simplex virus type 2 pUL21 phosphorylation impairs secondary envelopment of cytoplasmic nucleocapsids.

It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

Functions of the UL51 protein during the herpesvirus life cycle.

The herpesvirus UL51 protein is a multifunctional tegument protein involved in the regulation of multiple aspects of the viral life cycle. This article reviews the biological characteristics of the UL51 protein and its functions in herpesviruses, including participating in the maintenance of the viral assembly complex (cVAC) during viral assembly, affecting the production of mature viral particles and promoting primary and secondary envelopment, as well as its positive impact on viral cell-to-cell spread (CCS) through interactions with multiple viral proteins and its key role in the proliferation and pathogenicity of the virus in the later stage of infection. This paper discusses how the UL51 protein participates in the life cycle of herpesviruses and provides new ideas for further research on UL51 protein function.

Complete genome of the new bacilliform virus that causes Milky Hemolymph Syndrome in Chionoecetes bairdi (Rathbun, 1924)

The genome of a new member of the Nimaviridae family has been sequenced. The Chionoecetes bairdi bacilliform virus (CbBV) causes Milky Hemolymph Syndrome (MHS) in Chionoecetes bairdi populations of the Pacific coast of Kamchatka. The CbBV genome is represented by double-stranded DNA with a length of 245,567 nucleotides containing 120 ORFs. Of these, 85 proteins had significant matches in the NCBI database, and 57 genes encoded capsid, envelope, tegument and nonstructural proteins. Comparative analysis of the genomes of CbBV and a number of representatives of the class nuclear arthropod large DNA viruses (NALDVs) made it possible to isolate 49 evolutionarily conserved orthologue core genes. Among them, 5 were multicopy genes, and 44 were single-copy genes. There were ancestral genes characteristic of all Naldaviricetes − per os infectivity complex genes, one DNA polymerase gene and one thymidylate synthase gene.Phylogenetic analysis of representatives of the Nimaviridae family revealed that the CbBV and Chionoecetes opilio bacilliform virus (CoBV) form an independent clade within the family separate from the clade containing WSSV strains. This is supported by data on the order and arrangement of genes in the genomes of nimaviruses that were identical within each clade but differed between them. In addition, a high identity of the genomes and proteomes of CbBV and CoBV (approximately 99%) was shown, and their identity with WSSV strains was no more than 33%.The data on the structure of the genome of the new virus that causes MHS in C. bairdi indicate that it belongs to the family Nimaviridae, genus Whispovirus. Thus, the CbBV infecting the commercially important species of Tanner crab in populations of the Pacific coast of Kamchatka is the second “wild” representative of replicating nimaviruses whose genome has been characterized after the CoBV that causes MHS in C. opilio in populations of the Sea of Japan. The discovery of a new member of the family that infects decapods indicates the prevalence of nimaviruses in marine ecosystems. The information obtained is important for understanding the evolution of representatives of the class of nuclear arthropod large DNA viruses. The discovery of a new nimavirus that causes MHS in Chionoecetes crabs, in contrast to the white spot syndrome (WSS) caused by WSSV strains, makes it relevant to identify two variants and possibly species within the family, namely, WSSV and Milky Hemolymph Syndrome virus (MHSV).

Transmission patterns of malignant catarrhal fever in sheep and cattle in Karnataka, India.

Malignant catarrhal fever (MCF) presents a sporadic yet significant threat to livestock and wildlife. A comprehensive investigation in Karnataka, India into the prevalence and transmission patterns of sheep-associated MCF (SA-MCF) was conducted. A total of 507 sheep peripheral blood leukocyte samples from 13 districts along with 27 cows and 10 buffalo samples from various regions in Karnataka were tested for SA-MCF infection i.e. Ovine gammaherpesvirus 2 (OvHV-2) using heminested PCR. Furthermore, serum samples collected from 73 cows and 15 buffalo suspected of MCF were tested using a commercially available ELISA kit. Additionally, histopathological examinations of affected tissues and phylogenetic analysis of viral tegument protein sequences were conducted. Our findings indicated a 20.11%, 33.33% and 20% positivity for OvHV-2 in sheep, cows and buffalo respectively by PCR. Statistical analysis revealed a significant association between the age of sheep and the detection of OvHV-2. Seven cows and one buffalo serum samples tested positive for ELISA. Clinical findings in bovids were consistent with typical MCF signs, and histopathological results revealed multi-organ involvement characterised by necrotising vasculitis and lymphoid hyperplasia. The nucleotide pairwise identity matrix revealed 99.5% identity between the sequences obtained in the study with sequences from other states. The phylogenetic analysis of partial tegument protein sequences from bovid and sheep samples suggested a close genetic relationship between the local OvHV-2 strains and those from various global regions. Crucially, this study underscores the widespread presence of SA-MCF in Karnataka, with significant implications for both livestock management and wildlife conservation.

Identification of a novel neurovirulence factor encoded by the cryptic orphan gene UL31.6 of herpes simplex virus 1.

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.

Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane.

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.

The precise function of alphaherpesvirus tegument proteins and their interactions during the viral life cycle.

Alphaherpesvirus is a widespread pathogen that causes diverse diseases in humans and animals and can severely damage host health. Alphaherpesvirus particles comprise a DNA core, capsid, tegument and envelope; the tegument is located between the nuclear capsid and envelope. According to biochemical and proteomic analyses of alphaherpesvirus particles, the tegument contains at least 24 viral proteins and plays an important role in the alphaherpesvirus life cycle. This article reviews the important role of tegument proteins and their interactions during the viral life cycle to provide a reference and inspiration for understanding alphaherpesvirus infection pathogenesis and identifying new antiviral strategies.

Pseudorabies virus tegument protein US2 antagonizes antiviral innate immunity by targeting cGAS-STING signaling pathway.

The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

Subclinical Ovine Gammaherpesvirus 2-Related Infections in Free-Ranging Wild Boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2), is a Macavirus and the cause of sheep-associated malignant catarrhal fever (SA-MCF), in which sheep are the asymptomatic reservoir hosts. Susceptible mammalian populations infected by OvGHV2 may develop clinical SA-MCF or subclinical infections. All members of the Macavirus genus known to be associated with MCF are collectively referred to as the MCF virus (MCFV) complex. This report describes the occurrence of subclinical OvGHV2-related infections in free-ranging wild boars (Sus scrofa) from southern Brazil. Specific body organs (n = 14) and biological samples (nasal and oral swabs; n = 17) were collected from 24 asymptomatic wild boars from a conservation unit located within the Central-eastern mesoregion of Paraná State. Organs were processed to observe histopathological patterns suggestive of diseases of domestic animals; only pulmonary samples were used in an immunohistochemical assay designed to detect MCFV tissue antigens. Furthermore, all samples were submitted to molecular assays designed to detect the OvGHV2 tegument protein gene. Viral-induced pneumonia was diagnosed in two wild boars; one of these contained OvGHV2 DNA, with MCFV antigens identified in the other. Additionally, MCFV tissue antigens were detected within pulmonary epithelial cells of the lungs with and without pulmonary disease. Collectively, OvGHV2 was detected in 37.5% (9/24) of all wild boars, with detection occurring in the organs of 57.1% (8/14) wild boars and the oral cavity of one animal. These results demonstrated that these wild boars were subclinically infected by OvGHV2, and that infection produced typical pulmonary alterations. In addition, the detection of OvGHV2 within the oral cavity of one wild boar may suggest that this animal may be a potential disseminator of this pathogen to susceptible animal populations, including livestock and wildlife, acting as a possible bridge host for OvGHV2. Furthermore, infection by OvGHV2 probably occurred due to incidental contact with asymptomatic sheep maintained within the surrounding rural areas and not within the conservation units.