In Vero monkey cells and HEp-2 human epidermoid carcinoma cells infected with herpes simplex virus 1 the proteins (COP, galactosyltransferase, and a-mannosidase II associated with the Golgi apparatus appear to be associated with numerous smaller structures dispersed throughout the cytoplasm. Concomitantly, the intracytoplasmic ligands oflectins normally associated wholly (Helirpomatia

Postmitotic neurons need to keep their cell cycle under control to survive and maintain a differentiated state. This study aims to test the hypothesis that the chemokine CXCL12 regulates neuronal survival and differentiation by promoting Rb function, as suggested by previous studies showing that CXCL12 protects neurons from apoptosis induced by Rb loss. To this end, the effect of CXCL12 on Rb expression and transcriptional activity and the role of Rb in CXCL12-induced neuronal survival were studied. CXCL12 increases Rb protein and RNA levels in rat cortical neurons. The chemokine also stimulates an exogenous Rb promoter expressed in these neurons and counteracts the inhibition of the Rb promoter induced by E2F1 overexpression. Furthermore CXCL12 stimulates Rb activity as a transcription repressor. The effects of CXCL12 are mediated by its specific receptor CXCR4, and do not require the presence of glia. Finally, shRNA studies show that Rb expression is crucial to the neuroprotective activity of CXCL12 as indicated by NMDA-neurotoxicity assays. These findings suggest that proper CXCR4 stimulation in the mature CNS can prevent impairment of the Rb-E2F pathway and support neuronal survival. This is important to maintain CNS integrity in physiological conditions and prevent neuronal injury and loss typical of many neurodegenerative and neuroinflammatory conditions. Cell Death and Differentiation ( 1,2 Stimulation of CXCR4 by its endogenous ligand leads to activation of intracellular pathways affecting neuronal survival, migration, and neurotransmission.1,3 For instance, CXCR4 stimulates the PI3K/Akt pathway and regulates cell-cycle proteins in neurons. [3][4][5][6] Under pathological conditions at least some of these essential CXCR4 functions are compromised, leading to neuronal dysfunction/death. 1,7 Thus, a complete understanding of the effects of CXCR4 activation in the brain has important physiological and pathological implications.Rb is a well-known transcriptional repressor, which controls cell-cycle progression, differentiation/survival, and genomic integrity. Rb is also implicated in fundamental CNS developmental processes such as, neuronal migration, differentiation, and neurite extension. 8,9 The effects of Rb on cell survival are primarily due to regulation of members of the E2F family of transcription factors, though Rb can also directly inactivate proapoptotic proteins, such as pp32.10 E2F proteins stimulate expression of genes promoting cell-cycle progression in proliferating cells, whereas they induce apoptotic genes in differentiated cells, including neurons.11 Rb is phosphorylated at several phosphoacceptor sites by specific kinases. 12 Hence, the immediate effect of Rb phosphorylation is to remove Rb from its specific promoters, leading to inhibition of Rb-dependent gene repression. Rb phosphorylation results in enhanced E2F1 transcriptional activity culminating in death of postmitotic neurons.11 Alterations of Rb/E2F pathway were reported in various neuropathologies, such as Parkinson's and Alzhei...

In uninfected cells the G 2 /M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U L 13 and ␣22/U S 1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.The studies described in this report stemmed from the observation that the infected cell protein No.0 (ICP0) of herpes simplex virus 1 (HSV-1) binds to and stabilizes cyclin D3 (18). Further studies led to the observation that ICP0 and cyclin D3 colocalize in the infected cell nuclei and that ICP0 does not interfere with the phosphorylation of retinoblastoma protein (pRb) by cyclin D3-cdk4 complex. A role for cyclin D3 in the biology of HSV-1 emerged from mapping studies (44). Thus, substitution of aspartic acid 199 with alanine in ICP0 abolished stabilization of cyclin D3, reduced the yields of virus from resting cells, and reduced the capacity of the virus to invade the mouse central nervous system from a peripheral site. These studies demonstrated that HSV requires the participation of cell cycle proteins in the course of its replication even though the virus replicates efficiently in both resting and dividing cells. This conclusion is also supported by other observations, although in most instances a direct link to viral proteins is not yet available. Thus, pRb and p53 have been detected in the replication ...

Abstract-Many membranous organelles and protein complexes are normally transported anterograde within axons to the presynaptic terminal, and details of the motors, adaptors and cargoes have received significant attention. Much less is known about the transport in neurons of non-membrane bound particles, such as mRNAs and their associated proteins. We propose that herpes simplex virus type 1 (HSV) can be used to study the detailed mechanisms regulating long distance transport of particles in axons. A critical step in the transmission of HSV from one infected neuron to the next is the polarized anterograde axonal transport of viral DNA from the host infected nerve cell body to the axon terminal. Using the in vivo mouse retinal ganglion cell model infected with wild type virus or a mutant strain that lacks the protein Us9, we found that Us9 protein was necessary for long distance anterograde axonal transport of viral nucleocapsid (DNA surrounded by capsid proteins), but unnecessary for transport of virus envelope. Thus, we conclude that nucleocapsid can be transported independently down axons via a Us9-dependent mechanism.