Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing.

Genome sequencing and assembly of viral genomes within the Herpesviridae family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb. We have optimized DNA extraction and sequencing with ONT to capture the whole genome of HSV-1 as a single read. Although previous studies described the presence of four different genome isomers of HSV, we provided the first report on capturing all four variants' full-length genome as single reads. These isomers were found to be present in almost equal proportion in the sequenced DNA preparation. IMPORTANCE With the advent of next-generation sequencing platforms, genome sequencing of viruses can be performed in a relatively shorter time frame in even the most austere conditions. Ultralong read sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to capture the full-length genome of DNA viruses as a single read. By optimizing ONT for this purpose, we captured the genome (~154 Kb) of a clinical strain of herpes simplex virus 1 (HSV-1). Additionally, we captured full-length sequences of the four isomers of lab-grown HSV-1 virus and were able to determine the frequency of each within the isogenic population. This method will open new directions in studying the significance of these isomers and their clinical relevance to HSV-1 infections. It will also improve basic studies on the recombination and replication of this virus.

A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity.

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HAPR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI+), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HAPR8 This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/ß receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HAPR8 Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase.

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a "naked" state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication.

As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity.

Herpes simplex virus 1 Us3 deletion mutant is infective despite impaired capsid translocation to the cytoplasm.

Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i) The number of R7041(∆US3) capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii) The mean number of R7041(∆US3) virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii) 98% of R7041(∆US3) virions were in the perinuclear space; (iv) The number of R7041(∆US3) capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3) yields were 2.37×10(8) and HSV-1 yields 1.57×10(8) PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3) virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Multifluorescence live analysis of herpes simplex virus type-1 replication.

The possibility to label specific viral and cellular structures with live cell markers such as autofluorescent proteins has greatly contributed to our understanding of diverse steps of the virus life cycle, as it allows monitoring virus replication in a spatial and temporal fashion. Here, we describe the multi-fluorescent analysis of the multi-compartment herpes simplex virus type-1 by live-cell confocal laser scanning microscopy.

Neuromyelitis optica: a challenging diagnosis at secondary hospital.

Known since the 19th century, neuromyelitis optica (NMO), or Devic's disease, is an idiopathic immune-mediated inflammatory demyelinating disease of the central nervous system selectively affecting the optic nerve and spinal cord. Commonly diagnosed in demyelinating diseases reference centers, we report an 18-year-old female patient who sought medical attention with a 3-month history of weight loss, headache, and vomiting, followed by diplopia, a burning sensation over the lower limbs, and difficulty walking. A few days prior to hospital admission, the muscle strength in her lower limbs became worse and ascended to the upper limbs associated with sensory changes in the trunk and voiding dysfunction. At admission, the neurological examination was consistent with a spinal cord syndrome. After few days of hospitalization, she was tetraplegic with severe signs of brainstem involvement requiring mechanical ventilatory support. Intravenous methylprednisolone and cyclophosphamide were promptly started after ruling out the diagnosis of infectious disease and cord compression. Due to no substantial early improvement, intravenous immunoglobulin was also used. From then on, the neurological status gradually improved. Magnetic resonance imaging showed extensive demyelinating features in the spinal cord, and the serum IgG autoantibody was negative. The patient was referred to a tertiary neurological reference center where she remains under treatment.

The herpes simplex virus 1 U(S)3 regulates phospholipid synthesis.

Herpes simplex virus type 1 capsids bud at nuclear and Golgi membranes for envelopment by phospholipid bilayers. In the absence of U(S)3, nuclear membranes form multiple folds harboring virions that suggests disturbance in membrane turnover. Therefore, we investigated phospholipid metabolism in cells infected with the U(S)3 deletion mutant R7041(ΔU(S)3), and quantified membranes involved in viral envelopment. We report that (i) [(3)H]-choline incorporation into nuclear membranes and cytoplasmic membranes was enhanced peaking at 12 or 20 h post inoculation with wild type HSV-1 and R7041(ΔU(S)3), respectively, (ii) the surface area of nuclear membranes increased until 24 h of R7041(ΔU(S)3) infection forming folds that equaled ~45% of the nuclear surface, (iii) the surface area of viral envelopes between nuclear membranes equaled ~2400 R7041(ΔU(S)3) virions per cell, and (iv) during R7041(ΔU(S)3) infection, the Golgi complex expanded dramatically. The data indicate that U(S)3 plays a significant role in regulation of membrane biosynthesis.

Herpes simplex virus 1 induces de novo phospholipid synthesis.

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [³H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [³H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.

Herpes simplex virus type 1/adeno-associated virus hybrid vectors.

Herpes simplex virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size up to 150 kbp and, therefore, allow extensive combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other viruses to create hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other viruses that either direct integration of transgene sequences into the host genome or allow episomal maintenance of the vector. Thus, the advantages of the HSV-1 amplicon system, including large transgene capacity, broad host range, strong nuclear localization, and availability of helper virus-free packaging systems are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA. Adeno-associated virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The AAV rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that contain the rep gene and a transgene cassette flanked by AAV ITRs. These HSV/AAV hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression.

Inhibition of herpes simplex virus type 1 replication by adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase activities.

Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.

Live visualization of herpes simplex virus type 1 compartment dynamics.

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.