RESUMO
The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927-5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.
Assuntos
Herpesvirus Humano 1/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Alanina/metabolismo , Animais , Capsídeo/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 4/metabolismo , Mutação/genética , Fenótipo , Tirosina/metabolismo , Células Vero , Vírion/metabolismoRESUMO
Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115-6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2, engineered not to express either UL11 or gM, replicated more than 1 log unit less efficiently and produced significantly smaller plaques than UL11-null or gM-null viruses alone, in agreement with the results of Leege et al. (T. Leege et al., J. Virol. 83:896-907, 2009). Analyses of particle-to-PFU ratios, relative plaque size, and kinetics of virus growth and ultrastructural visualization of glycoprotein-deficient mutant and wild-type virions indicate that gDΔct, gE, and gM function in a cooperative but not redundant manner in infectious virion morphogenesis. Overall, comparisons of single, double, and triple mutant viruses generated in the same HSV-1(F) genetic background indicated that lack of either UL20 or gK expression caused the most severe defects in cytoplasmic envelopment, egress, and infectious virus production, followed by the double deletion of UL11 and gM.
Assuntos
Citoplasma/metabolismo , Glicoproteínas/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Liberação de Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Ordem dos Genes , Vetores Genéticos , Glicoproteínas/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/ultraestrutura , Humanos , Mutação , Fenótipo , Ligação Proteica , Proteoma/metabolismo , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
VG2025 is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) that uses transcriptional and translational dual regulation (TTDR) of critical viral genes to enhance virus safety and promote tumor-specific virus replication without reducing virulence. The TTDR platform is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor-specific carcinoembryonic antigen (CEA) promoter, coupled with translational control of the neurovirulence factor ICP34.5 using multiple microRNA (miR)-binding sites. VG2025 further incorporates IL-12 and the IL-15/IL-15 receptor alpha subunit complex to enhance the antitumor and immune stimulatory properties of oncolytic HSVs. The TTDR strategy was verified in vitro and shown to be highly selective. Strong in vivo antitumor efficacy was observed following both intratumoral and intravenous administration. Clear abscopal and immune memory effects were also evident, indicating a robust antitumor immune response. Gene expression profiling of treated tumors revealed increased immune cell infiltration and activation of multiple immune-signaling pathways when compared with the backbone virus. Absence of neurotoxicity was verified in mice and in rhesus monkeys. Taken together, the enhanced tumor clearance, excellent safety profile, and positive correlation between CEA levels and viral replication efficiency may provide an opportunity for using biomarker-based precision medicine in oncolytic virotherapy.
RESUMO
The development of effective cancer vaccines remains a significant challenge due to immune tolerance and limited clinical benefits. Oncolytic herpes simplex virus type 1 (oHSV-1) has shown promise as a cancer therapy, but efficacy is often limited in advanced cancers. In this study, we constructed and characterized a novel oHSV-1 virus (VG22401) expressing the human epidermal growth factor receptor 2 (HER2), a transmembrane glycoprotein overexpressed in many carcinomas. VG22401 exhibited efficient replication and HER2 payload expression in both human and mouse colorectal cancer cells. Mice immunized with VG22401 showed significant binding of serum anti-HER2 antibodies to HER2-expressing tumor cells, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, mice primed with VG22401 and intratumorally boosted with the same virus showed enhanced antitumor efficacy in a bilateral syngeneic HER2(+) tumor model, compared to HER2-null backbone virus. This effect was accompanied by the induction of anti-HER2 T cell responses. Our findings suggest that peripheral priming with HER2-expressing oHSV-1 followed by an intratumoral boost with the same virus can significantly enhance antitumor immunity and efficacy, presenting a promising strategy for cancer immunotherapy.
RESUMO
Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKDelta31-47, gKDelta31-68, and gKDelta31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKDelta31-47 and gKDelta31-117 exhibited a gK-null (DeltagK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKDelta31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKDelta31-47 and gKDelta31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBDelta28syn) caused extensive virus-induced cell fusion. However, the gBDelta28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKDelta31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKDelta31-68, DeltagK, or UL20-null virus. The gKa peptide complemented the gKDelta31-47 and gKDelta31-68 mutant viruses for infectious-virus production and for gKDelta31-68/gBDelta28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.
Assuntos
Fusão Celular , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Virais/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Teste de Complementação Genética , Herpesvirus Humano 1/genética , Deleção de Sequência , Proteínas do Envelope Viral/fisiologia , Ensaio de Placa Viral , Proteínas Virais/genéticaRESUMO
Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans-Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gDDeltact) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg+gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg+gDDeltact mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation codons of UL20 (UL20ctgctg). All recombinant viruses were constructed by using the double-Red, site-directed mutagenesis system implemented on the HSV-1(F) genome cloned into a bacterial artificial chromosome. The gEctg, gEctg+gMctg, gDDeltact, and gEctg+gDDeltact viruses produced viral plaques on African monkey kidney cells (Vero), as well as other cells, that were on average approximately 30 to 50% smaller than those produced by the wild-type virus HSV-1(F). In contrast, the UL20ctgctg virus produced very small plaques containing three to five cells, as reported previously for the DeltaUL20 virus lacking the entire UL20 gene. Viral replication kinetics of intracellular and extracellular viruses revealed that all recombinant viruses produced viral titers similar to those produced by the wild-type HSV-1(F) virus intracellularly and extracellularly at late times postinfection, with the exception of the UL20ctgctg and DeltaUL20 viruses, which replicated more than two-and-a-half logs less efficiently than HSV-1(F). Electron microscopy confirmed that all viruses, regardless of their different gene mutations, efficiently produced enveloped virions within infected cells, with the exception of the UL20ctgctg and DeltaUL20 viruses, which accumulated high levels of unenveloped virions in the cytoplasm. These results show that the carboxyl terminus of gD and the full-length gE, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Similarly, gM and gE do not function alone or in a redundant manner in cytoplasmic envelopment and virion egress, confirming previous findings.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas do Envelope Viral/genética , Montagem de Vírus , Animais , Chlorocebus aethiops , Citoplasma/virologia , DNA Viral/genética , Perfilação da Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/ultraestrutura , Humanos , Mutagênese Sítio-Dirigida , Mutação , Células Vero , Ensaio de Placa Viral , Proteínas Virais/genéticaRESUMO
Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.