A herpes simplex virus 1 (McKrae) mutant lacking the glycoprotein K gene is unable to infect via neuronal axons and egress from neuronal cell bodies.

UNLABELLED: We have shown that the herpes simplex virus 1 (HSV-1) gK gene is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglion neuroinvasion in mice (A. T. David, A. Baghian, T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, Curr. Eye Res. 33:455-467, 2008). To further investigate the role of gK in neuronal infection, we utilized a microfluidic chamber system separating neuronal cell bodies and axonal termini. HSV-1 (McKrae) engineered virus constitutively expressing enhanced green fluorescence protein (GFP) was efficiently transmitted in both a retrograde and an anterograde manner. These results were corroborated by expression of virion structural proteins in either chamber, as well as detection of viral genomes and infectious viruses. In contrast, efficient infection of either chamber with a gK-null virus did not result in infection of the apposed chamber. These results show that gK is an important determinant in virion axonal infection. Moreover, the inability of the gK-null virus to be transmitted in an anterograde manner suggests that virions acquire cytoplasmic envelopes prior to entering axons. IMPORTANCE: Herpes simplex virus 1 (HSV-1) enters mucosal epithelial cells and neurons via fusion of the viral envelope with cellular membranes, mediated by viral glycoprotein B (gB) in cooperation with other viral glycoproteins. Retrograde transport of virions to neuronal cell bodies (somata) establishes lifelong latent infection in ganglionic neurons. We have previously reported that gK binds gB and is required for gB-mediated membrane fusion (Jambunatathan et al., J. Virol. 85:12910-12918, 2011; V. N. Chouljenko, A. V. Iyer, S. Chowdhury, J. Kim, and K. G. Kousoulas, J. Virol. 84:8596-8606, 2010). In the current study, we constructed a recombinant virus with the gK gene deleted in the highly virulent ocular HSV-1 strain McKrae. This recombinant virus failed to infect rat ganglionic neuronal axons alone or cocultured with Vero cells in microfluidic chambers. In addition, lack of gK expression prevented anterograde transmission of virions. These results suggest that gK is a critical determinant for neuronal infection and transmission.

Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice.

BACKGROUND: The NV1020 oncolytic herpes simplex virus type-1 has shown significant promise for the treatment of many different types of tumors in experimental animal models and human trials. Previously, we described the construction and use of the NV1020-like virus OncSyn to treat human breast tumors implanted in nude mice. The syncytial mutation gKsyn1 (Ala-to-Val at position 40) was introduced into the OncSyn viral genome cloned into a bacterial artificial chromosome using double-red mutagenesis in E. coli to produce the OncdSyn virus carrying syncytial mutations in both gB(syn3) and gK(syn1). RESULTS: The OncdSyn virus caused extensive virus-induced cell fusion in cell culture. The oncolytic potential of the OncSyn and OncdSyn viruses was tested in the highly metastatic syngeneic mouse model system, which utilizes 4T1 murine mammary cancer cells implanted within the interscapular region of Balb/c mice. Mice were given three consecutive intratumor injections of OncSyn, OncdSyn, or phosphate buffered saline four days apart. Both OncSyn and OncdSyn virus injections resulted in significant reduction of tumor sizes (p < 0.05) compared to control tumors. Virus treated mice but not controls showed a marked reduction of metastatic foci in lungs and internal organs. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.296). CONCLUSION: These results show that the attenuated, but highly fusogenic OncSyn and OncdSyn viruses can effectively reduce primary and metastatic breast tumors in immuncompetent mice. The available bac-cloned OncSyn and OncdSyn viral genomes can be rapidly modified to express a number of different anti-tumor and immunomodulatory genes that can further enhance their anti-tumor potency.

The herpes simplex virus type 1 (HSV-1) glycoprotein K(gK) is essential for viral corneal spread and neuroinvasiveness.

PURPOSE: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice. METHODS: The recombinant virus HSV-1 (McKrae) Delta gK (MKDelta gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKDelta gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored. RESULTS: Mice infected with the MKDelta gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKDelta gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKDelta gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKDelta gK. CONCLUSIONS: The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKDelta gK inoculated mice.