The Herpes Simplex Virus Type 1 Locus That Encodes
http://www.100md.com
病菌学杂志 2005年第22期
Department of Veterinary and Biomedical Sciences, Nebraska Center for Virology, University of Nebraska, Fair Street at East Campus Loop, Lincoln, Nebraska 68583-0905
Department of Ophthalmology, College of Medicine, University of California at Irvine, UCIMC, Bldg. 55, Room 204, 101 The City Drive, Orange, California 92868-4380-02
School of Biological Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska
ABSTRACT
Herpes simplex virus type 1 (HSV-1) is the leading cause of virus-induced encephalitis; however, the viral genes that regulate encephalitis have not been well characterized. In this study, we tested whether the LAT (latency-associated transcript) locus regulates the frequency of encephalitis in male or female mice. Male BALB/c mice are more susceptible to HSV-1-induced encephalitis than age-matched female BALB/c mice. Deletion of LAT coding sequences reduced the frequency of encephalitis. A recombinant virus containing the first 1.5 kb of the LAT coding sequence induces levels of encephalitis in male BALB/c mice similar to those induced by wild-type HSV-1.
TEXT
Approximately 90% of the population is infected with herpes simplex virus type 1 (HSV-1) (29, 55). Following acute infection, latency is established in sensory neurons in trigeminal ganglia or sacral dorsal root ganglia (19, 50). Reactivation from latency periodically occurs, resulting in virus transmission and recurrent disease (19, 20).
The latency-associated transcript (LAT) is abundantly transcribed in latently infected neurons (8, 10, 11, 22, 27, 44, 48, 51, 52). An 8.3-kb LAT transcript is spliced, yielding the stable 2-kb LAT intron that localizes to the nucleus (2, 31, 49) and an unstable 6.3-kb transcript (11, 44, 57). Proper splicing of LAT is necessary for establishment and maintenance of latency (21). The HSV-1 McKrae strain is frequently shed in tears of latently infected rabbits because of spontaneous reactivation (39-43), and LAT gene expression is crucial for spontaneous reactivation. LAT inhibits apoptosis in cultured cells and trigeminal ganglia of infected rabbits or mice (1, 14, 17, 18, 21, 37). The ability of LAT to interfere with apoptosis correlates with spontaneous reactivation (17, 18).
HSV-1-induced encephalitis is a severe form of focal necrotizing encephalitis that affects approximately 2,000 individuals each year in the United States (12, 24, 54, 55). Without antiviral therapy, the mortality rate can be 70% (45, 46). Approximately 2/3 of HSV-1-induced encephalitis cases are due to reactivation from latency (56); consequently, high morbidity and long-term complications occur despite antiviral treatment (23, 26, 45).
In general, females induce a stronger immune response because they express higher levels of lymphocytes and cytokines (3, 5, 16). Susceptibilities to encephalomyocarditis virus (9), vesicular stomatitis virus (4), coxsackievirus B3 (15), and HSV-1 infections in mice (13) differ based on gender. Furthermore, a HSV-2 vaccine trial in humans succeeded in females but not in males (47). In female mice, a HSV resistance locus (sex modifier locus) has been identified (25). HSV-1 hematogenous infection is more pathogenic to females (6), and estrogen influences pseudorabies virus infections in the central nervous system (53), suggesting that gender-specific factors, male as well as female, influence host-pathogen interactions.
Based on the observations that gender can influence the pathogenic potential of certain viruses, we tested whether LAT influences the frequencies of encephalitis in male and female mice. Adult female or male BALB/c mice were infected with the wild-type (wt) McKrae strain or the LAT locus mutant, dLAT2903 (Fig. 1). As previously described for 129 Sv//Ev or RGKO male mice (13), male BALB/c mice infected with wt HSV-1 or dLAT2903R (rescued dLAT2903) (Fig. 1A) had frequencies of survival significantly lower than those of female BALB/c mice (Fig. 2A and B). A Tukey-Kramer multiple-comparison post-analysis of variance test revealed that survival rates for male and female mice infected with wt HSV-1 or dLAT2903R were significantly different (P = 0.004). Male mice infected with dLAT2903 had significantly higher (P = 0.003) levels of survival than those infected with wt HSV-1 or dLAT2903R. As previously reported (38), female mice infected with dLAT2903, wt HSV-1, or dLAT2903R exhibited similar survival rates.
The deletion within dLAT2903 prevents expression of at least four genetic elements (Fig. 1C): (i) the stable 2-kb LAT; (ii) the first 1.5 kb of LAT, which contains only part of the stable 2-kb LAT and is sufficient for high levels of spontaneous reactivation in the rabbit eye model (41) or for trigeminal ganglia explant-induced reactivation in mice (38); (iii) the AL transcript (36); and (iv) the UOL transcript (30). To localize regions of LAT necessary for enhancing encephalitis in male BALB/c mice, we tested LAT3.3A (Fig. 1D) and LAT1.8A (Fig. 1E). LAT3.3A expresses UOL, AL, and one copy of the first 1.5 kb of LAT but does not express the intact stable 2-kb LAT. LAT1.8A contains one copy of the minimal UOL coding sequences but not AL or LAT. Male, but not female, BALB/c mice infected with LAT3.3A had a frequency of encephalitis significantly higher than that for age-matched male mice infected with LAT1.8A (P = 0.003) (Fig. 2A and B). Furthermore, LAT3.3A, wt HSV-1, and dLAT2903R induced similar levels of encephalitis in male BALB/c mice, indicating that expression of the intact stable 2-kb LAT does not regulate gender-specific encephalitis. Finally, LAT1.8A induced levels of encephalitis in male BALB/c mice similar to those induced by dLAT2903.
Following infection of male or female mice with the respective viruses, the mice that died typically succumbed to fatal encephalitis between 7 and 10 days after infection (Fig. 2C). Similar virus titers were also detected in brain homogenates at 2, 4, or 8 days after infection, regardless of the virus used to infect male (Fig. 3) or female BALB/c mice (data not shown). Consistent with previous studies (28, 35, 38), we also found similar levels of infectious virus in ocular swabs from male and female mice following infection with the virus strains. These studies suggested that the reduced frequency of encephalitis in male BALB/c mice infected with dLAT2903 was not due to reduced viral titers in the brain.
Since the LAT locus inhibits interferon (IFN) RNA expression in neuronal cell types (34), we tested whether IFN was differentially expressed in brains of mice during acute infection (2 to 8 days after infection) or after latency was established (14 days after infection). Using primers that specifically amplify mouse gamma IFN (IFN-) (Fig. 4C), male mice infected with dLAT2903 contained readily detectable levels of IFN- RNA at 6, 8, and 14 days after infection, but mock-infected mice did not (Fig. 4A). In contrast, male mice infected with the McKrae strain or dLAT2903R expressed IFN- RNA at 6 and 8 days, but not at 14 days, after infection. Viral glycoprotein (gC) was detected on days 6 and 8 regardless of the virus used for infection. At 4 days after infection, low levels of gC RNA were detected in male mice infected with the McKrae strain or with dLAT2903R but not in those infected with dLAT2903. gC RNA expression levels were similar in brains of male mice at 6 and 8 days after infection, regardless of which virus was used. As expected, LAT was not detected in mice infected with dLAT2903 (Fig. 4A). Female mice infected with dLAT2903, dLAT2903R, or wt HSV-1 expressed IFN- RNA 6 and 8 days after infection (Fig. 4B). These findings suggested that a correlation exists between survival of male mice infected with dLAT2903 and prolonged IFN- RNA expression in brains. Our results are also consistent with studies demonstrating that IFN- protects against HSV infection (7, 13).
In summary, these studies indicated that the ability of HSV-1 to induce high levels of fatal encephalitis in male BALB/c mice was dependent on expression of the first 1.5 kb of LAT, expression of UOL, and/or expression of AL. We further suggest that the male mouse model may be useful for identifying sequences in the LAT locus that regulate the latency-reactivation cycle and neuropathogenesis. The first 1.5 kb of LAT promotes spontaneous reactivation (40, 41) and inhibits caspase 8-induced apoptosis efficiently (33). The functions of AL and UOL are not known. Additional studies are required to prove whether AL, UOL, or the 1.5-kb LAT sequence regulates neurovirulence in male BALB/c mice. Since expressions of the 1.5-kb LAT, of UOL, and of AL are regulated by the same promoter and their coding sequences overlap one another, preparing recombinant viruses that express just one of the respective gene products is a challenge.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant 1P20RR15635 and by two USDA grants (2002-35204 and 2003-02213).
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Department of Ophthalmology, College of Medicine, University of California at Irvine, UCIMC, Bldg. 55, Room 204, 101 The City Drive, Orange, California 92868-4380-02
School of Biological Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska
ABSTRACT
Herpes simplex virus type 1 (HSV-1) is the leading cause of virus-induced encephalitis; however, the viral genes that regulate encephalitis have not been well characterized. In this study, we tested whether the LAT (latency-associated transcript) locus regulates the frequency of encephalitis in male or female mice. Male BALB/c mice are more susceptible to HSV-1-induced encephalitis than age-matched female BALB/c mice. Deletion of LAT coding sequences reduced the frequency of encephalitis. A recombinant virus containing the first 1.5 kb of the LAT coding sequence induces levels of encephalitis in male BALB/c mice similar to those induced by wild-type HSV-1.
TEXT
Approximately 90% of the population is infected with herpes simplex virus type 1 (HSV-1) (29, 55). Following acute infection, latency is established in sensory neurons in trigeminal ganglia or sacral dorsal root ganglia (19, 50). Reactivation from latency periodically occurs, resulting in virus transmission and recurrent disease (19, 20).
The latency-associated transcript (LAT) is abundantly transcribed in latently infected neurons (8, 10, 11, 22, 27, 44, 48, 51, 52). An 8.3-kb LAT transcript is spliced, yielding the stable 2-kb LAT intron that localizes to the nucleus (2, 31, 49) and an unstable 6.3-kb transcript (11, 44, 57). Proper splicing of LAT is necessary for establishment and maintenance of latency (21). The HSV-1 McKrae strain is frequently shed in tears of latently infected rabbits because of spontaneous reactivation (39-43), and LAT gene expression is crucial for spontaneous reactivation. LAT inhibits apoptosis in cultured cells and trigeminal ganglia of infected rabbits or mice (1, 14, 17, 18, 21, 37). The ability of LAT to interfere with apoptosis correlates with spontaneous reactivation (17, 18).
HSV-1-induced encephalitis is a severe form of focal necrotizing encephalitis that affects approximately 2,000 individuals each year in the United States (12, 24, 54, 55). Without antiviral therapy, the mortality rate can be 70% (45, 46). Approximately 2/3 of HSV-1-induced encephalitis cases are due to reactivation from latency (56); consequently, high morbidity and long-term complications occur despite antiviral treatment (23, 26, 45).
In general, females induce a stronger immune response because they express higher levels of lymphocytes and cytokines (3, 5, 16). Susceptibilities to encephalomyocarditis virus (9), vesicular stomatitis virus (4), coxsackievirus B3 (15), and HSV-1 infections in mice (13) differ based on gender. Furthermore, a HSV-2 vaccine trial in humans succeeded in females but not in males (47). In female mice, a HSV resistance locus (sex modifier locus) has been identified (25). HSV-1 hematogenous infection is more pathogenic to females (6), and estrogen influences pseudorabies virus infections in the central nervous system (53), suggesting that gender-specific factors, male as well as female, influence host-pathogen interactions.
Based on the observations that gender can influence the pathogenic potential of certain viruses, we tested whether LAT influences the frequencies of encephalitis in male and female mice. Adult female or male BALB/c mice were infected with the wild-type (wt) McKrae strain or the LAT locus mutant, dLAT2903 (Fig. 1). As previously described for 129 Sv//Ev or RGKO male mice (13), male BALB/c mice infected with wt HSV-1 or dLAT2903R (rescued dLAT2903) (Fig. 1A) had frequencies of survival significantly lower than those of female BALB/c mice (Fig. 2A and B). A Tukey-Kramer multiple-comparison post-analysis of variance test revealed that survival rates for male and female mice infected with wt HSV-1 or dLAT2903R were significantly different (P = 0.004). Male mice infected with dLAT2903 had significantly higher (P = 0.003) levels of survival than those infected with wt HSV-1 or dLAT2903R. As previously reported (38), female mice infected with dLAT2903, wt HSV-1, or dLAT2903R exhibited similar survival rates.
The deletion within dLAT2903 prevents expression of at least four genetic elements (Fig. 1C): (i) the stable 2-kb LAT; (ii) the first 1.5 kb of LAT, which contains only part of the stable 2-kb LAT and is sufficient for high levels of spontaneous reactivation in the rabbit eye model (41) or for trigeminal ganglia explant-induced reactivation in mice (38); (iii) the AL transcript (36); and (iv) the UOL transcript (30). To localize regions of LAT necessary for enhancing encephalitis in male BALB/c mice, we tested LAT3.3A (Fig. 1D) and LAT1.8A (Fig. 1E). LAT3.3A expresses UOL, AL, and one copy of the first 1.5 kb of LAT but does not express the intact stable 2-kb LAT. LAT1.8A contains one copy of the minimal UOL coding sequences but not AL or LAT. Male, but not female, BALB/c mice infected with LAT3.3A had a frequency of encephalitis significantly higher than that for age-matched male mice infected with LAT1.8A (P = 0.003) (Fig. 2A and B). Furthermore, LAT3.3A, wt HSV-1, and dLAT2903R induced similar levels of encephalitis in male BALB/c mice, indicating that expression of the intact stable 2-kb LAT does not regulate gender-specific encephalitis. Finally, LAT1.8A induced levels of encephalitis in male BALB/c mice similar to those induced by dLAT2903.
Following infection of male or female mice with the respective viruses, the mice that died typically succumbed to fatal encephalitis between 7 and 10 days after infection (Fig. 2C). Similar virus titers were also detected in brain homogenates at 2, 4, or 8 days after infection, regardless of the virus used to infect male (Fig. 3) or female BALB/c mice (data not shown). Consistent with previous studies (28, 35, 38), we also found similar levels of infectious virus in ocular swabs from male and female mice following infection with the virus strains. These studies suggested that the reduced frequency of encephalitis in male BALB/c mice infected with dLAT2903 was not due to reduced viral titers in the brain.
Since the LAT locus inhibits interferon (IFN) RNA expression in neuronal cell types (34), we tested whether IFN was differentially expressed in brains of mice during acute infection (2 to 8 days after infection) or after latency was established (14 days after infection). Using primers that specifically amplify mouse gamma IFN (IFN-) (Fig. 4C), male mice infected with dLAT2903 contained readily detectable levels of IFN- RNA at 6, 8, and 14 days after infection, but mock-infected mice did not (Fig. 4A). In contrast, male mice infected with the McKrae strain or dLAT2903R expressed IFN- RNA at 6 and 8 days, but not at 14 days, after infection. Viral glycoprotein (gC) was detected on days 6 and 8 regardless of the virus used for infection. At 4 days after infection, low levels of gC RNA were detected in male mice infected with the McKrae strain or with dLAT2903R but not in those infected with dLAT2903. gC RNA expression levels were similar in brains of male mice at 6 and 8 days after infection, regardless of which virus was used. As expected, LAT was not detected in mice infected with dLAT2903 (Fig. 4A). Female mice infected with dLAT2903, dLAT2903R, or wt HSV-1 expressed IFN- RNA 6 and 8 days after infection (Fig. 4B). These findings suggested that a correlation exists between survival of male mice infected with dLAT2903 and prolonged IFN- RNA expression in brains. Our results are also consistent with studies demonstrating that IFN- protects against HSV infection (7, 13).
In summary, these studies indicated that the ability of HSV-1 to induce high levels of fatal encephalitis in male BALB/c mice was dependent on expression of the first 1.5 kb of LAT, expression of UOL, and/or expression of AL. We further suggest that the male mouse model may be useful for identifying sequences in the LAT locus that regulate the latency-reactivation cycle and neuropathogenesis. The first 1.5 kb of LAT promotes spontaneous reactivation (40, 41) and inhibits caspase 8-induced apoptosis efficiently (33). The functions of AL and UOL are not known. Additional studies are required to prove whether AL, UOL, or the 1.5-kb LAT sequence regulates neurovirulence in male BALB/c mice. Since expressions of the 1.5-kb LAT, of UOL, and of AL are regulated by the same promoter and their coding sequences overlap one another, preparing recombinant viruses that express just one of the respective gene products is a challenge.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant 1P20RR15635 and by two USDA grants (2002-35204 and 2003-02213).
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