Varicella-zoster virus gene 66 transcription and translation in latently infected human Ganglia

Abstract

Latent infection with varicella-zoster virus (VZV) is characterized by restricted virus gene expression and the absence of virus production. Of the approximately 70 predicted VZV genes, only five (genes 4, 21, 29, 62, and 63) have been shown by multiple techniques to be transcribed during latency. IE62, the protein product of VZV gene 62, is the major immediate-early (IE) virus-encoded transactivator of viral gene transcription and plays a pivotal role in transactivating viral genes during lytic infection. The protein kinase (66-pk) encoded by VZV gene 66 phosphorylates IE62, resulting in cytoplasmic accumulation of IE62 that mitigates nuclear IE62-induced gene activation. Analysis of latently infected human trigeminal ganglia for 66-pk expression by reverse transcriptase-dependent nested PCR, including DNA sequence analysis, in situ hybridization, and immunohistochemistry, revealed VZV open reading frame 66 to be a previously unrecognized latently expressed virus gene and suggests that prevention of IE62 import to the nucleus by VZV 66-pk phosphorylation is one possible mechanism by which VZV latency is maintained.

FIG. 1.

PCR amplification of VZV cDNA. (A) Total TG RNA extracted was reverse transcribed with (lane 1) or without (lane 2) RT. In a second reaction, VZV-infected MeWo cell RNA was reverse transcribed with (lane 3) or without (lane 4) RT. Each cDNA product was amplified with primers designed to yield products spanning the poly(A) tract. Amplified products were resolved by agarose gel electrophoresis and stained with ethidium bromide (left). VZV-specific amplification products were transferred to nylon membranes and probed with ³²P-end-labeled, internally located oligonucleotides (right). Both TG and VZV-infected MeWo cell cDNA contained actin transcripts (top gels); VZV gene 40 transcripts were detected only in virus-infected MeWo cell cDNA (middle gels); and VZV gene 66 transcripts were detected in TG and VZV-infected MeWo cell cDNA (bottom gels). In all instances, amplification was RT dependent (lanes 1 and 3). No product was seen when RT (lanes 2 and 4) or template DNA (lane 5) was omitted from the PCR. M, double-stranded DNA size markers. (B) Primer efficiencies were determined by PCR amplification of serial log₁₀ dilutions of VZV ORF 40 and ORF 66 artificial transcripts (AT). In the primary PCR, both ORF primer sets gave strong signals with 10⁸ copies of AT. In nested PCR, the ORF 40 primer set detected 10⁴ to 10⁵ copies of AT, while ORF 66 primers detected 10⁶ to 10⁷ of added AT. ND, no AT template DNA added to the PCR.

FIG. 2.

Detection of VZV transcripts by ISH. Adjacent sections of a latently infected human TG were hybridized to digoxigenin-labeled VZV-specific probes (66F1, 66R1, and 62-AS; Table 1). (A and B) No signal was detected after hybridization with a sense VZV gene 66 probe (A), whereas a strong signal was seen in the nuclei of two neurons (open arrows) and a weaker signal was seen in two neurons (dark arrows) after hybridization with an antisense VZV gene 66 probe (B). (C) A strong signal in three neurons (open arrows) was also detected after hybridization with an antisense probe to VZV gene 62. Magnification, ×450.

FIG. 3.

Immunohistochemical detection of VZV ORF 66 protein. TG sections from two subjects were incubated with antibody to VZV gene 66 protein as described in Materials and Methods. (A and B) Strong labeling in the cytoplasm of one neuron (open arrow) and weaker labeling in another neuron (closed arrow) from the first subject was detected (A), whereas no signal was detected in the second subject (B). TG sections from the first subject but not the second subject were positive for VZV gene 66 transcripts by ISH (data not shown). (C and D) VZV gene 66 protein was present in VZV-infected MeWo cells (C), but not in uninfected MeWo cells (D). Images were captured at a magnification of ×427.5; panel B was printed at a magnification of ×541.5.