Fluorescence tagging of herpes simplex virus type 1 proteins by mutagenesis of a bacterial artificial chromosome

[no abstract

The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain

Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral herpesviralmutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17(+)) Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment

Early, Active, and Specific Localization of Herpes Simplex Virus Type 1 gM to Nuclear Membranes▿

Thirteen different glycoproteins are incorporated into mature herpes simplex virus type 1 (HSV-1) virions. Five of them play important roles during entry, while others intervene during egress of the virus. Although HSV-1 gM is not essential in cell culture, its deletion reduces viral yields and promotes syncytium formation. Furthermore, gM is conserved among herpesviruses, is essential for several of them, and can redirect the gD and gH/gL viral glycoproteins from the cell surface to the trans-Golgi network, where gM presumably modulates final capsid envelopment. Late in infection, gM reaches the nuclear envelope and decorates perinuclear virions. This process seemingly requires UL31 and UL34 and occurs when several markers of the trans-Golgi network have relocalized to the nucleus. However, the precise mechanism of gM nuclear targeting is unclear. We now report that gM is quickly and specifically targeted to nuclear membranes in a virus-dependent manner. This occurs prior to the HSV-1-induced reorganization of the trans-Golgi network and before gM enters the secretory pathway. The presence of a high-mannose glycosylation pattern on gM further corroborated these findings. While gM was targeted to the inner nuclear membrane early in infection, its partners gD, gH, gN, VP22, UL31, and UL34 did not colocalize with gM. These data suggest that nuclear gM fulfills an early nuclear function that is independent of its known interaction partners and its function in viral egress

A herpes simplex virus-derived replicative vector expressing LIF limits experimental demyelinating disease and modulates autoimmunity.

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17(+))-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE

Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17+)▿

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17+)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17+)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17+)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes

Nuclear aggregates induced by HSV1-XFPVP26 impair nuclear capsid egress.

<p>Vero cells were infected (inf.) with 10 PFU/cell of HSV1(17⁺)blueLox (A, wild type), HSV1(17⁺)blueLox-mRFPVP26_{Δaa<b>1</b>–7} (B), or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7}(C), and fixed at 9 h with PFA. Alternatively, cells were transfected (transf.) with pHSV1(17⁺)blueLox-GFPVP26_{Δaa<b>5</b>–7} (D) or pHSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7} (E), and fixed at 24 h. In addition to the intrinsic fluorescence of the XFPVP26 constructs (mRFPVP26 or GFPVP26), the subcellular localization of VP26 (α-VP26) and VP5 (MAb 5C10) were analyzed after permeabilization with TX-100 and immunolabeling by confocal fluorescence microscopy. The nuclei were stained with TO-PRO-3 (A, E). Arrows highlight cytoplasmic capsids (A–D) or incoming capsids at the nuclear rim of a neighboring cell (Ei). Scale bar: 10 µm.</p

Quantification of nuclear aggregate formation.

<p>Vero cells were infected with 10 PFU/cell of HSV1(17⁺)blueLox (B) or HSV1(17⁺)blueLox-GFPVP26_{Δaa<b>1</b>–7}-gDmRFP (C), and the cells were fixed at 4, 6, 8, 10, or 12 h. After permeabilization, HSV1(17⁺)blueLox infected cells were labeled with an antibody directed against VP26. According to their intranuclear VP26 phenotype, the cells were classified into “none”, “single”, “grainy” and “aggregate” (A). The numbers above the columns describe the number of nuclei analyzed for each time point.</p

HSV1-VP26 constructs.

<p>(A) 1^st column: HSV1 constructs in which the SCP VP26 has been tagged with different fluorescent protein domains. 2^nd column: Genomic organization of the UL35 region approximately drawn to scale. The gene UL35 coding for VP26 has been disrupted by replacing it with lacZ or an rpsLneo cassette out of frame. Some constructs lack a 65 bp region upstream of UL35 (D65 bp) including the first seven N-terminal codons of VP26 (Daa1–7), while others lack only four (Δaa1–4) or just one (Daa1) codon. For the present study, the fluorescent protein tag was inserted between VP26 residues 4 and 8 (Daa5–7). Due to the mutagenesis, some strains contain additional linkers (*, AW; **, NSS; ***, HST). 3^rd column: Propensity of the fluorescent protein (FP) to dimerize <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Espagne1" target="_blank">[76]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044177#pone.0044177-Campbell1" target="_blank">[80]</a>. 4^th column: Ability of the construct to replicate and to form plaques (+++, similar to wild type; ++ attenuated, but robust growth; − strongly attenuated, tiny plaques; −−, single fluorescent cells, no plaques). 5^th column: Propensity of the construct to induce nuclear aggregates (+++, large irregular shaped aggregates; ++, large aggregates early after infection or transfection; +, aggregates late in infection; − aggregates in less than 2% of cells even late in infection). 6^th column: References. (B) Nucleotide (upper lines) and amino acid (lower lines) sequences of the UL34/UL35 (pUL34/VP26) intergenic region. The 3¢ end of the UL34 ORF until the 5¢ start of the UL35 ORF are shown for wild type HSV-1, the GFPVP26_{Äaa<b>5</b>–7} (Äaa5–7) and GFPVP26_{Äaa<b>1</b>–7} (Äaa1–7) mutants. Additional nucleotides inserted during mutagenesis are shown in bold capitals, and the GFP amino acids are shown in italics. Putative Inr late promoter elements are underlined with the element perfectly matching the consensus sequence being underlined and in italics. The original amino acids encoded by UL35 are shown in bold capitals, the inserted GFP residues in italic capitals and the additional linker residues in normal script capitals.</p