Amita Patel

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

Novel Less-Abundant Viral MicroRNAs Encoded by Herpes Simplex Virus 2 Latency-Associated Transcript and Their Roles in Regulating ICP34.5 and ICP0 mRNAs

ABSTRACT We recently identified an acutely and latently expressed viral microRNA (miRNA), miR-I, encoded by herpes simplex virus 2 (HSV-2) latency-associated transcript (LAT) through small RNA cloning and two miRNAs encoded by HSV-1 LAT through prediction. We now report the use of high-throughput sequencing technology to identify two additional relatively less-abundant viral miRNAs, miR-II and miR-III, encoded by HSV-2 LAT exon 2. miR-II includes two miRNAs, miR-II-5p and miR-II-3p, which are processed from the same miRNA precursor. miR-II and miR-III map antisense to the 5′ untranslated region of ICP34.5 and to the coding region of ICP0 exon 3, respectively. These novel miRNAs are conserved in different HSV-2 strains, and their presence in infected- and transfected-cell cultures was confirmed by Northern hybridization. All three HSV-2 LAT-encoded miRNAs map to genome locations similar to those of three out of four identified HSV-1 LAT-encoded miRNAs, but the sequences of these miRNAs are not conserved. The expression of LAT-encoded miRNAs is negatively regulated by ICP4, the major viral transactivator. We further show that, similar to miR-I, miR-II is able to efficiently silence the expression of ICP34.5, a key viral neurovirulence factor, and that miR-III is able to silence the expression of ICP0, a key viral transactivator. All these data suggest that LAT sequences likely contribute to HSV latency and reactivation through tight control of these LAT-encoded miRNAs and their viral targets.

Development of resistance to acyclovir during chronic infection with the Oka vaccine strain of varicella-zoster virus, in an immunosuppressed child.

A 1-year-old boy was vaccinated with the Oka strain of varicella just prior to the discovery of a tumor that required intensive antitumor therapy. Three months later he developed herpes zoster, which developed into chronic verrucous lesions that were refractory to treatment with acyclovir and which subsequently disseminated. DNA from a biopsy specimen of a chronic herpes-zoster lesion indicated that the Oka vaccine strain of the the virus caused this severe complication. Analysis of this viral DNA demonstrated a mutation in the viral thymidine kinase gene. Plasmids containing this altered gene were unable to produce functional thymidine kinase in an in vitro translation system. The presence of this mutation would explain the clinical resistance to acyclovir. This is the first report of Oka-strain varicella virus causing severe disease after reactivation and of resistance to acyclovir during an infection caused by this virus.

Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine

High-potency inactivated poliovirus vaccine (eIPV) was combined with diphtheria-tetanus-pertussis (DTP) vaccine containing thimerosal as a preservative to simulate the performance of a potential tetravalent vaccine. Neither type 1 nor type 3 poliovirus antigens appeared to be affected by thimerosal after exposure for 1 h at 37 degrees C as measured by enzyme-linked immunosorbent assay (ELISA). One epitope on the type 2 antigen was damaged within 5 min of exposure; however, the overall potency was unchanged when measured using a polyclonal antibody preparation. Exposure to thimerosal at 37 degrees C decreased the potency of all three poliovirus types to well below the level caused by heat deterioration alone in 1-2 days and to 0% after 16-17 days. At 25 degrees C, the potency of type 1 poliovirus decreased by 46% in 1 day, whereas poliovirus types 2 and 3 were stable for 1 week. Storage of eIPV at 4 degrees C in the presence of thimerosal reduced the potency of type 1 poliovirus antigen to undetectable levels after 4-6 months. Type 2 and 3 antigens were less markedly affected by 8 months of exposure to thimerosal at 4 degrees C. The loss of potency of type 1 as measured by ELISA was paralleled by a reduced level of neutralizing antibodies in mice injected with these preparations. The results obtained from testing eIPV in combination with DTP and thimerosal were generally similar to those obtained using eIPV with thimerosal. It remains to be seen to what extent thimerosal will affect the immunogenicity of eIPV in humans when injected as combined eIPV-DTP vaccine.

Herpes simplex virus 2 microRNA miR-H6 is a novel latency-associated transcript-associated microRNA, but reduction of its expression does not influence the establishment of viral latency or the recurrence phenotype.

ABSTRACT The herpes simplex virus 2 (HSV-2) viral microRNA (miRNA) designated miR-H6 is located upstream of the latency-associated transcript (LAT) promoter region on the strand opposite the LAT. Deletion of the LAT promoter and part of LAT exon 1 abolished HSV-2 miR-H6 expression in acutely and latently infected guinea pig dorsal root ganglia (DRG), suggesting that this region is needed both for the expression of LAT-encoded miRNAs and for miR-H6 expression in vivo. Relative to cells infected with a viral rescuant, miR-H6 expression was significantly reduced in cells infected with a mutant HSV-2 virus, NotPolyA, with an insertion of a simian virus (SV40) polyadenylation signal sequence between the LAT promoter and miR-H6 sequences. In addition, expression of miR-H6, but not LAT or viral DNA, was significantly reduced in both mouse trigeminal ganglia (TG) and guinea pig DRG latently infected with the NotPolyA mutant. Guinea pigs infected with NotPolyA experienced reduced neurological complications of acute infection relative to those infected with the rescuant, but the recurrence phenotype of the NotPolyA mutant was similar to those of its rescuant and wild-type HSV-2, indicating that reduction of miR-H6 expression is not by itself able to alter the establishment of latency for the wild-type virus or the recurrence phenotype. Furthermore, the mutation in NotPolyA did not affect the propensity of wild-type HSV-2 to establish latency in neurons positive for subtype marker KH10. In contrast to published reports regarding its HSV-1 homolog, HSV-2 miR-H6 did not affect ICP4 expression in transfected or infected cells. We hypothesize that viral miRNAs associated with LAT expression are likely to work collectively, contributing to the phenotype attributed to the LAT.

Herpes Simplex Virus Latency-Associated Transcript Sequence Downstream of the Promoter Influences Type-Specific Reactivation and Viral Neurotropism

ABSTRACT Herpes simplex virus (HSV) establishes latency in sensory nerve ganglia during acute infection and may later periodically reactivate to cause recurrent disease. HSV type 1 (HSV-1) reactivates more efficiently than HSV-2 from trigeminal ganglia while HSV-2 reactivates more efficiently than HSV-1 from lumbosacral dorsal root ganglia (DRG) to cause recurrent orofacial and genital herpes, respectively. In a previous study, a chimeric HSV-2 that expressed the latency-associated transcript (LAT) from HSV-1 reactivated similarly to wild-type HSV-1, suggesting that the LAT influences the type-specific reactivation phenotype of HSV-2. To further define the LAT region essential for type-specific reactivation, we constructed additional chimeric HSV-2 viruses by replacing the HSV-2 LAT promoter (HSV2-LAT-P1) or 2.5 kb of the HSV-2 LAT sequence (HSV2-LAT-S1) with the corresponding regions from HSV-1. HSV2-LAT-S1 was impaired for reactivation in the guinea pig genital model, while its rescuant and HSV2-LAT-P1 reactivated with a wild-type HSV-2 phenotype. Moreover, recurrences of HSV-2-LAT-S1 were frequently fatal, in contrast to the relatively mild recurrences of the other viruses. During recurrences, HSV2-LAT-S1 DNA increased more in the sacral cord compared to its rescuant or HSV-2. Thus, the LAT sequence region, not the LAT promoter region, provides essential elements for type-specific reactivation of HSV-2 and also plays a role in viral neurotropism. HSV-1 DNA, as quantified by real-time PCR, was more abundant in the lumbar spinal cord, while HSV-2 DNA was more abundant in the sacral spinal cord, which may provide insights into the mechanism for type-specific reactivation and different patterns of central nervous system infection of HSV-1 and HSV-2.

Latency-Associated Transcript (LAT) Exon 1 Controls Herpes Simplex Virus Species-Specific Phenotypes: Reactivation in the Guinea Pig Genital Model and Neuron Subtype-Specific Latent Expression of LAT

ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2 cause similar acute infections but differ in their abilities to reactivate from trigeminal and lumbosacral dorsal root ganglia. During latency, HSV-1 and HSV-2 also preferentially express their latency-associated transcripts (LATs) in different sensory neuronal subtypes that are positive for A5 and KH10 markers, respectively. Chimeric virus studies showed that LAT region sequences influence both of these viral species-specific phenotypes. To further map the LAT region sequences responsible for these phenotypes, we constructed the chimeric virus HSV2-LAT-E1, in which exon 1 (from the LAT TATA to the intron splice site) was replaced by the corresponding sequence from HSV-1 LAT. In intravaginally infected guinea pigs, HSV2-LAT-E1 reactivated inefficiently relative to the efficiency of its rescuant and wild-type HSV-2, but it yielded similar levels of viral DNA, LAT, and ICP0 during acute and latent infection. HSV2-LAT-E1 preferentially expressed the LAT in A5+ neurons (as does HSV-1), while the chimeric viruses HSV2-LAT-P1 (LAT promoter swap) and HSV2-LAT-S1 (LAT sequence swap downstream of the promoter) exhibited neuron subtype-specific latent LAT expression phenotypes more similar to that of HSV-2 than that of HSV-1. Rescuant viruses displayed the wild-type HSV-2 phenotypes of efficient reactivation in the guinea pig genital model and a tendency to express LAT in KH10+ neurons. The region that is critical for HSV species-specific differences in latency and reactivation thus lies between the LAT TATA and the intron splice site, and minor differences in the 5′ ends of chimeric sequences in HSV2-LAT-E1 and HSV2-LAT-S1 point to sequences immediately downstream of the LAT TATA.

Universal virus detection by degenerate-oligonucleotide primed polymerase chain reaction of purified viral nucleic acids

Summary This study describes a novel non-specific universal virus detection method that permits molecular detection of viruses in biological materials containing mixtures of cells and viruses. Samples are subjected to nuclease digestion and ultracentrifugation to separate encapsidated viral nucleic acids from cellular nucleic acids. A degenerate oligonucleotide primer PCR (DOP-PCR) that has been optimized for the non-specific amplification of virus sized genomes is then employed. Virus identification is performed by sequencing of cloned DOP-PCR products followed by sequence comparison to sequences published in GenBank. This method was used to detect a variety of DNA viruses (including HSV, VZV, SV40, AAV, and EBV) and RNA viruses (including HTLV-I, HTLV-II, influenza, and poliovirus), which were spiked into cells, constitutively expressed in cell culture, or detected in productively infected cultured cells. This novel approach was compared with a non-specific virus detection method used previously and found to be several logs more sensitive. This type of approach has potential utility in solving virus detection and discovery problems where other methods have failed.

Herpes Simplex Virus 2 Expresses a Novel Form of ICP34.5, a Major Viral Neurovirulence Factor, through Regulated Alternative Splicing

ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2, two closely related neurotropic human herpesviruses, achieve neurotropism through ICP34.5, a major viral neurovirulence factor. In this report, in addition to the full-length 38-kDa protein (ICP34.5α), we identified a 28-kDa novel form of ICP34.5 (ICP34.5β) in HSV-2-infected cells. ICP34.5β is translated from unspliced ICP34.5 mRNA, with the retained intron introducing a premature stop codon. Thus, ICP34.5β lacks the C-terminal conserved GADD34 domain but includes 19 additional amino acids encoded by the intron. Although a fraction of both HSV-2 ICP34.5 proteins are detected in the nucleolus, ICP34.5α is predominantly located in cytoplasm, and ICP34.5β is mainly detected more diffusely in the nucleus. ICP34.5β is unable to counteract PKR-mediated eIF2 phosphorylation but does not interfere with ICP34.5αs function in this process. Efficient expression of ICP34.5β in cell culture assays is dependent on viral infection or expression of ICP27, a multifunctional immediate-early gene. The effect of ICP27 on the ICP34.5β protein level is attributed to its selective inhibition of ICP34.5 splicing, which results in increased expression of ICP34.5β but a reduced level of ICP34.5α. The C- terminal KH3 domain but not the RNA binding domain of ICP27 is required for its specific inhibition of ICP34.5 splicing and promotion of ICP34.5β expression. Our results suggest that the expression of ICP34.5α and ICP34.5β is tightly regulated in HSV-2 and likely contributes to viral pathogenesis.

Spread of Herpes Simplex Virus to the Spinal Cord Is Independent of Spread to Dorsal Root Ganglia

ABSTRACT Levels of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in dorsal root ganglia (DRG) and spinal cord (SC) were quantified after inoculation of guinea pig genitals and footpads. In genital infection, viral DNA reached SC and DRG simultaneously (at 2 to 3 days after inoculation) but was more abundant in SC than in DRG. After inoculation of footpads, which lack parasympathetic innervation, the viruses spread more efficiently to DRG than to SC. These results show important differences between genital and footpad infections, including independence of spread to DRG and SC, and imply that autonomic neurons may play an important role in the pathogenesis of viral latency after genital inoculation.