Genetics of Herpes Simplex Virus Type-1 tegument proteins involved in virion morphogenesis and egress

Herpes simplex virus type-1 (HSV-1) morphogenesis occurs in multiple stages within infected cells. Initially, the virion capsid assembles within the nucleus and buds through the nuclear membrane into the cytoplasm. Within the cytoplasm, additional tegument proteins attach to the capsid and the fully tegumented capsids bud into trans-Golgi network (TGN) derived vesicles. Enveloped virions are ultimately secreted to extracellular spaces. The process by which the cytoplasmic capsids bud into TGN-derived vesicles is not well understood. The prevalent model calls for specific interactions among viral tegument proteins and membrane proteins and glycoproteins embedded within TGN membranes. To further investigate the roles of tegument proteins in cytoplasmic virion envelopment, we constructed deletion mutants of UL11, UL20, both UL11 and UL20, and UL16. UL11 is involved in cytoplasmic virion envelopment. The ΔUL11 virus exhibits large amounts of unenveloped capsids in the cytoplasm of infected cells. The phenotype of the double null virus most closely resembled that of the UL20 single null virus (ΔUL20) in all areas: plaque phenotype, growth kinetics, and ultrastructural characteristics. To asses whether UL11 has any affect on UL20/gK localization, confocal experiments to determine the localization of UL11, UL20 and gK were undertaken, revealing that UL11 transport was completely independent of UL20/gK. Taken together these results indicate that UL11 acts at a step in cytoplasmic envelopment downstream of UL20, and UL20 is required for proper UL11 function. However, UL11 is not dependent upon the UL20/gK heterodimer for its transport. To assess the role of UL16 in virion morphogenesis and egress, the YEbac102ΔUL16 virus was constructed using a recently described RED markerless recombination system. ΔUL16 showed a large accumulation of intranuclear capsids not seen in the ΔUL11 virus. This result indicates a two-fold role for UL16 in virion morphogenesis and egress: 1) The nuclear accumulation of capsids seems to suggest that the first and most important role of UL16 is in intranuclear capsid assembly/egress. 2) The cytoplasmic accumulation of capsids suggests that UL16 also plays a role in cytoplasmic envelopment. These results indicate a possible pathway for the juxtaposition of cytoplasmic capsids with TGN-derived vesicles for final cytoplasmic envelopment

Susceptibility of Bacillus anthracis to Gamma and Cherry bacteriophage

Bacillus anthracis is a bacterium that causes severe disease mainly in ruminants, but can affect any mammal, including humans. A popular method for the detection of this organism is susceptibility of the bacterial isolate to g bacteriophage. However, to date no study on the resistance of a wide variety of B. anthracis isolates has been conducted. The following study examines the rate of resistance of a wide range of B. anthracis isolates to g phage as well as another phage specific for B. anthracis known as Cherry phage. We also compared susceptibility to phage with another detection method, susceptibility to penicillin, to determine any association between the two. The origin of the resistant isolates was examined to determine associations between resistance and isolate origin. Finally, the gross structure and resistant rates of the two phages were compared to determine any relation between the two viruses. We found that B. anthracis showed 20% resistance to g phage, which we propose is too high to continue its use as a reliable diagnostic tool. No association was found between resistance to penicillin and resistance to phage. No association was found between isolate origin and resistance. No conclusions could be drawn as to the relationship between the two phages

The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment

The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion

Laboratory growth of denitrifying water column microbial consortia from deep-sea shipwrecks in the northern Gulf of Mexico [version 3; referees: 2 approved]

Background: Shipwrecks serve as a rich source for novel microbial populations that have largely remained undiscovered. Low temperatures, lack of sunlight, and the availability of substrates derived from the shipwreck’s hull and cargo may provide an environment in which microbes can develop unique metabolic adaptations.   Methods: To test our hypothesis that shipwrecks could influence the microbial population involved in denitrification when a consortium is grown in the laboratory, we collected samples proximate to two steel shipwrecks in the northern Gulf of Mexico. Then under laboratory conditions, we grew two independent denitrifying microbial consortia. Each consortium was grown by using the BART assay system and analyzed based on growth kinetics, ion chromatography and 16S amplicon sequencing. Results: Both denitrifying consortia were different from each other based on varied growth profiles, rates of nitrate utilization and 16S amplicon sequencing. Conclusions: Our observations conclude that the laboratory grown water column microbial consortia from deep-sea shipwrecks in the Gulf of Mexico are able to undergo aggressive denitrification

Laboratory growth of denitrifying water column microbial consortia from deep-sea shipwrecks in the northern Gulf of Mexico [version 2; referees: 2 approved]

Background: Shipwrecks serve as a rich source for novel microbial populations that have largely remained undiscovered. Low temperatures, lack of sunlight, and the availability of substrates derived from the shipwreck’s hull and cargo may provide an environment in which microbes can develop unique metabolic adaptations.   Methods: To test our hypothesis that shipwrecks could influence the microbial population involved in denitrification when a consortium is grown in the laboratory, we collected samples proximate to two steel shipwrecks in the northern Gulf of Mexico. Then under laboratory conditions, we grew two independent denitrifying microbial consortia. Each consortium was grown by using the BART assay system and analyzed based on growth kinetics, ion chromatography and 16S amplicon sequencing. Results: Both denitrifying consortia were different from each other based on varied growth profiles, rates of nitrate utilization and 16S amplicon sequencing. Conclusions: Our observations conclude that the laboratory grown water column microbial consortia from deep-sea shipwrecks in the Gulf of Mexico are able to undergo aggressive denitrification

UL20 Protein Functions Precede and Are Required for the UL11 Functions of Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment

Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20- and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment

The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment-2

<p><b>Copyright information:</b></p><p>Taken from "The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment"</p><p>http://www.virologyj.com/content/4/1/120</p><p>Virology Journal 2007;4():120-120.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2186317.</p><p></p>K) or gB(syn3) mutation. Viral plaques were visualized by immunohistochemistry at 24 hpi

The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment-5

<p><b>Copyright information:</b></p><p>Taken from "The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment"</p><p>http://www.virologyj.com/content/4/1/120</p><p>Virology Journal 2007;4():120-120.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2186317.</p><p></p>live conditions with aV5 (gK) mAb for 6 hours. Cells were washed thoroughly, fixed, and processed for confocal microscopy. After permeabilization, antibodies a3 × FLAG, aV5 and aTGN46 were used to identify, UL20p, gK and TGN46, respectively

The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment-1

<p><b>Copyright information:</b></p><p>Taken from "The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment"</p><p>http://www.virologyj.com/content/4/1/120</p><p>Virology Journal 2007;4():120-120.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2186317.</p><p></p>d at 24 hours post infection and tittered on Vero cells (see Materials & Methods). The error bars shown represent the maximum and minimum complementation ratios obtained from three independent experiments, and the bar height represents the average complementation ratio