out of other people's pubs and believing that you've assimilated a complete
theory! IT IS SUCH A DISSERVICE TO OTHERS!

Yes there are people that are genius enough to be medical practitioners
and virologists that aren't licensed.

****YOU ARE CLEARLY NOT ONE OF THEM PERL VON MOLSON!!!****

*ALL* glycoproteins are translated from HOST RNA on the rough endoplasmic
reticulum then transported to the golgi body in vesicles to continue the
glycosylation process.  The glycoproteins are then transported in vesicles
to the nuclear or plasma membrane.

ABSTRACT

Inhibitory effects of ethanolic extracts from 10 Chinese herbs on
herpes simplex virus type 1 (HSV-1) replication were investigated. By a
bioassay-guided fractionation procedure, samarangenin B (Sam B) was
isolated from Limonium sinense; Sam B significantly suppressed HSV-1
multiplication in Vero cells without apparent cytotoxicity.
Time-of-addition experiments suggested that the inhibitory action of
Sam B on HSV-1 replication was not due to the blocking of virus
adsorption. In an attempt to further localize the point in the HSV-1
replication cycle where arrest occurred, a set of key regulatory events
leading to viral multiplication was examined, including viral
immediate-early ({alpha}), early (ß), and late ({gamma}) gene
expression and DNA replication. Results indicated that levels of
glycoprotein B (gB), gC, gD, gG, and infected-cell protein 5 (ICP5)
expression and gB mRNA expression in Vero cells were impeded by Sam B.
Data from PCR showed that replication of HSV-1 DNA in Vero cells was
arrested by Sam B. Furthermore, Sam B decreased DNA polymerase, ICP0,
and ICP4 gene expression in Vero cells. Results of an electrophoretic
mobility shift assay demonstrated that Sam B interrupted the formation
of an {alpha}-trans-induction factor/C1/Oct-1/GARAT multiprotein
complex. The mechanisms of antiviral action of Sam B seem to be
mediated, at least in part, by inhibiting HSV-1 {alpha} gene
expression, including expression of the ICP0 and ICP4 genes, by
blocking ß transcripts such as DNA polymerase mRNA, and by arresting
HSV-1 DNA synthesis and structural protein expression in Vero cells.
These results show that Sam B is an antiviral agent against HSV-1
replication.

INTRODUCTION

Herpes simplex virus type 1 (HSV-1) is an enveloped DNA virus which
causes a variety of infections in humans. After primary infection,
HSV-1 establishes latency in sensory and autonomic neurons innervating
the mucosal tissues, where primary infection takes place, and is
reactivated by the proper stimulus to cause recurrence (35). The period
of recurrence is irregular (20). Immunocompromised individuals and
those with cancer are in danger of recurrent HSV-1 infections (20, 21,
27). The recipients of organ transplantation are at high risk for
increased severity of HSV-1 infection (33). Infection with HSV-1 can
lead to life-threatening encephalitis and ocular infections that result
in corneal inflammation and scarification. This scarification is a
major cause of blindness in developing countries (8). In addition,
HSV-1 has been shown to be a factor for spreading human
immunodeficiency virus and causes severe diseases in AIDS patients (5,
29).

One successful replication cycle of HSV-1 is dependent on the
completion of a number of steps, including virion entry, subsequent
expression of viral immediate-early ({alpha}) genes such as
infected-cell protein 0 (ICP0) and ICP4 genes, early (ß1 and ß2)
genes including DNA polymerase and thymidine kinase genes, and late
({gamma}1 and {gamma}2) genes encoding glycoprotein B (gB), ICP5, and
gC, and unpaired DNA replication (35). The initial expression of HSV-1
{alpha} genes depends on the binding of the {alpha} trans-induction
factor ({alpha}TIF)/C1/Oct-1 multiprotein complex to the TAAGARAT (R,
purine; GARAT) sequences of the cis-acting site (31). Inhibition of any
of these stages blocks HSV-1 replication. Nucleoside analogues have
been extensively investigated in the search for effective
antiherpesvirus agents (9). Among these acyclovir is widely used for
the systemic treatment of HSV infections. It is a highly selective
antiviral agent because it is specifically phosphorylated by viral
thymidine kinase in infected cells (10, 13). However,
acyclovir-resistant HSV infection in immunocompromised patients such as
transplant patients and patients with AIDS has recently been observed
(6, 19). Therefore, it is desirable to develop new anti-HSV agents that
substitute for or complement acyclovir.

Chinese herbs are potential sources of useful edible and medicinal
plants. They are expected to find use as functional foods because of
their various biological activities such as immunomodulatory and
antitumor functions (22, 23). More and more people in developing
countries utilize traditional medicine for their major primary health
care needs (11, 17). However, ethnopharmacology also provides
scientists with an alternative approach for the discovery of antiviral
agents. The polysaccharides (30), anthraquinones (39), triterpenes
(38), phloroglucinol (2), flavonoids (26), and catechin derivatives
(12) isolated from medicinal plants have been found to have inhibitory
activities against the replication of HSV-1. There has been a promising
result for a naturally occurring antiherpetic agent, n-docosanol, which
has recently completed extensive clinical evaluation and been approved
by the U.S. Food and Drug Administration as a topical treatment for
herpes labialis (1, 34, 36). These findings show that natural products
are still potential sources in the search for new antiherpetic agents.

In the present study, 10 Chinese herbs which are widely known in folk
medicine for the treatment of viral and bacterial infection were
selected for an anti-HSV-1 replication assay. The herbs were Ventilago
leiocarpa, Ecdysanthera rosea, Ecdysanthera utilis, Hippobroma
longiflora, Ardrisia brevicaulis, Selaginella delicatula, Limonium
sinense, Ardrisia japonica, Ardrisia violacea, and Andendron
benthamianum. The ethanolic extracts that showed appreciable anti-HSV-1
activity were separated by a bioassay-guided fractionation procedure.
The effect of active component samarangenin B (Sam B), isolated from L.
sinense, on HSV-1 {alpha}, ß, and {gamma} gene expression and DNA
replication in Vero cells was evaluated. The mechanisms of antiviral
action of Sam B were elucidated in vitro.

The plaque reduction assay offers a popular system to evaluate the
effect of antiviral agents against HSV-1 (3). In our study, 10 Chinese
herbs were screened by this model and L. sinense was found to contain
antiherpetic agents, supporting the validity of its use for
pharmacological studies. Results shown here indicated that Sam B
purified from L. sinense suppressed HSV-1 multiplication in Vero cells
without significantly reducing cell viability and growth. The
inhibitory effect of Sam B may be attributed to its interference with
structural proteins and DNA synthesis, DNA polymerase mRNA
transcription, and {alpha} gene expression of HSV-1. Hence, Sam B
suppression of viral replication might have important implications for
L. sinense therapeutic activity in microorganism infection. This is the
first report of the antiviral action mechanisms of Sam B.

Sam B isolated from L. sinense is a polyphenol flavonoid compound (26,
32). Results showing that Sam B impaired HSV-1 replication in Vero
cells were compatible with data reported by Vanden Berghe et al., which
indicate that many polyphenols are known for their antiherpesvirus
activities (40). Sam B blockage of HSV-1 replication was probably not
related to DMSO because cell viability and growth and HSV-1 replication
in Vero cells were not changed by DMSO. The morphology and
characteristics of Vero cells treated with Sam B or other Chinese herb
extracts were similar, suggesting that inhibitory effects of Sam B were
not related to the pH, osmolarity, or other physiology variables in
different preparations (data not shown). Although we did not determine
whether Sam B bound to virus, results of electron microscopy
observations indicated that the morphology of HSV-1 in the presence of
Sam B is unchanged from that in its absence (data not shown). A
comparison with control groups showed that the HSV-1 titer was not
significantly decreased when viral particles were treated with 25 µM
Sam B at 37°C for 1 h (data not shown). Moreover, pretreatment of
cells with Sam B or addition of the drug after viral adsorption
produced antiviral activity similar to that when HSV-1 and Sam B were
added at the same time. These results suggest that the binding of Sam B
to virion or host cells could not be a factor inhibiting virus
replication. The preliminary data indicated that pretreatment of Vero
cells with Sam B for 24 h and then its removal before infection still
inhibit virus yield (data not shown). It is possible that the binding
of Sam B to certain membrane molecules of the host cell different from
the receptor, resulting in interference with virus penetration into the
host cells, such as by interfering with virus-cell fusion, was not
related to the inhibitory action of Sam B.

In the host cells, HSV-1 replication is coordinately regulated and
sequentially ordered in a cascade and is believed to proceed as
follows: (i) {alpha}TIF, a {gamma} protein packaged in the virion,
turns on the {alpha} genes to be transcribed; (ii) expression of
{alpha} genes regulates the ß genes to be expressed; (iii) both
{alpha} and ß gene expression initiates HSV-1 DNA replication; (iv)
{gamma} genes are synthesized, and then virions are assembled; and (v)
HSV-1 is enveloped as it buds through the nuclear membrane (18, 28,
35). In the present study, we found that Sam B decreased ICP0 and ICP4
gene expression in Vero cells. We are currently attempting to further
elucidate whether the mechanisms by which Sam B suppresses ICP0 and
ICP4 gene expression are related to the initial transcriptional
transactivation events, including formation of
{alpha}TIF/C1/Oct-1/GARAT multiprotein complexes. Our initial
experiments using EMSA indicated that retarded species IEC has been
decreased in the GARAT probes incubated with Sam B-treated nuclear
extracts infected by HSV-1. Thus, the possibility that Sam B inhibited
HSV-1 replication through disturbance of {alpha}TIF/C1/Oct-1/GARAT
stable complex formation cannot be excluded. ICP0 has been reported to
perform several functions including selection of transcriptional
termination sites and stimulation of DNA synthesis. Although ICP0 is
not essential for HSV-1 replication in some cell cultures, defects in
the associated gene delay the expression of ß and {gamma} genes and
impair viral replication (35). ICP4 is the major transactivator of
HSV-1 genes. Thus, ICP4 and ICP0 play important roles in regulation of
ß and {gamma} gene expression and are essential for HSV-1 replication
(14). Moreover, the present data indicated that Sam B impaired DNA
polymerase transcripts and HSV-1 DNA synthesis in Vero cells. It is
known that a large array of proteins including DNA polymerase are
required for HSV-1 DNA synthesis. We suggest that the decrease in HSV-1
DNA synthesis due to Sam B is related to impairment of DNA polymerase.
Recently, we detected thymidine kinase mRNA expression in Vero cells by
RT-PCR, and preliminary results indicate that Sam B impeded thymidine
kinase mRNA expression (data not shown). We also proved that the
production of gB, gC, gD, gG, and ICP5 proteins in Vero cells was
attenuated by Sam B. ICP5 is a major capsid protein and is made both
early and late in infection. gB, gC, gD, and gG are all involved in the
viral envelope structure and play important roles in viral attachment
and penetration. The evidence demonstrates that ICP4 is required for
expression of {gamma} genes and that ICP4 binding sites enhance the
transcription of the gD gene in vitro (15). Although effects of Sam B
on gC, gD, gG, and ICP5 mRNA expression were not determined, it was
found that Sam B decreased gB mRNA expression in Vero cells. HSV-1 DNA
synthesis is required for gC mRNA expression. gD, gG, and ICP5 are
similar to gB in that their mRNA expression requires ICP4 proteins
(34). Thus, we predict that Sam B attenuates levels of gB, gC, gD, gG,
and ICP5 proteins in Vero cells, which may be related to impairment of
DNA synthesis and ICP4 and ICP0 production. The attenuation of DNA
synthesis and ICP4 and ICP0 production may cause decreases of gB, gC,
gD, gG, and ICP5 gene expression in Vero cells.

multiplication in Sam B-treated Vero cells, at least in part, was
related to (i) decreases in HSV-1 ICP0 and ICP4 gene expression due to
Sam B, which might be related to disturbance of the formation of
{alpha}TIF/C1/Oct-1/GARAT multiprotein complexes, (ii) reduction in DNA
polymerase transcriptions in the cells, (iii) inhibition of viral DNA
synthesis, (iv) interference with gB protein synthesis due to blockage
of gB mRNA synthesis, (v) impaired levels of viral capsid protein ICP5
and envelope proteins gC, gD, and gG, and (vi) no HSV-1 plaque
formation in Vero cells. Unlike dextran sulfate isolated from Sargassum
horneri, which inhibits HSV-1 replication at the adsorption step (16),
Sam B blocked HSV-1 replication at the immediate-early and early steps.
The expression of the immediate-early gene represents one stage of the
HSV-1 replication cycle that could be targeted by a novel antiviral
therapy to deliver a significant reduction in virus replication in both
acute and latent infections. The expression of the immediate-early gene
plays important roles in the regulation of all classes of viral genes
during lytic infection and is the key initiating event in the process
of reactivation of the latent HSV-1 genomes. Low levels of
immediate-early transcripts can be identified in latently infected
neuron cells. ICP0 mutant viruses that are defective in immediate-early
transactivation have been shown to reactivate very poorly from latent
infections (35). While nucleoside analogues have been successful in
treating acute infections, they fail to modulate reactivation of latent
virus. On the other hand, Sam B lacked the elevated cytotoxicity and
antiproliferative properties of interferons (4). Because Vero cells are
notoriously hardy and aneuploid, the effects of Sam B on the growth of
human peripheral blood mononuclear cells (PBMC), which are normal
diploid cells, were determined. Results indicated that Sam B did not
affect DNA, RNA, and protein synthesis in PBMC (data not shown) and
that the therapeutic index was about 13.2. Thus, small molecules
identified from Chinese herbs such as Sam B which act as inhibitors of
HSV-1 immediate-early gene expression may have the potential to impact
clinical disease to a far greater extent than currently marketed
nucleosides and cytokines. Future experiments with treatment of
HSV-1-infected animals with Sam B will be necessary to define whether
L. sinense can reduce experimental viral infection injury and prevent
recurrent HSV-1 infection. Moreover, this study not only demonstrates
that Chinese herbs are potential therapeutic drugs for the viral
infection but also supports a model for future protocol design in
preclinical studies.

http://aac.asm.org/cgi/content/full/46/9/2854

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=127446