Maryam Ahmed - Academia.edu (original) (raw)
Papers by Maryam Ahmed
Journal of Virology, 2001
During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral gene... more During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral genes is restricted to the latency-associated transcripts (LATs). The stable 2-kb LAT intron has been characterized previously and has been shown to accumulate to high levels in the nuclei of infected neurons. However, in productively infected tissue culture cells, this unique intron is also found in the cytoplasm. Although deletion mutant analysis has suggested that the region of the gene from which the intron is spliced plays a role in maintenance of latency or in reactivation from latency, no well-defined function has been ascribed specifically to the 2-kb LAT intron. Nevertheless, previous work has shown that it associates with 50S particles in the cytoplasm of acutely infected cells. Our studies tested the ability of the 2-kb LAT to dissociate from cytoplasmic protein complexes under various salt conditions. Results indicated that this association, which had been speculated to be mRNA-like, is actually more similar to the affinity of rRNAs for translational complexes. Furthermore, by immunoprecipitation analysis, we demonstrate that the 2-kb LAT associates with ribosomal as well as with splicing complexes in infected cells. Our results suggest that the 2-kb LAT is processed similarly to mRNAs in the nuclei of infected cells. However, in the cytoplasm, the 2-kb LAT may play a structural role in the ribosomal complex, similar to that of the cellular rRNAs, and therefore affect the functioning of the translational machinery.
Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression
Virology, 1997
In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatit... more In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression ...
Vesicular stomatitis virus as a treatment for colorectal cancer
Cancer Gene Therapy, 2011
M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the tr... more M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the treatment of metastatic colorectal cancer due to its ability to kill cancer cells that are defective in their antiviral responses. The oncolytic activity of recombinant wild-type and M protein mutant vesicular stomatitis viruses was determined in RKO, Hct116 and LoVo colorectal cancer cells, as well as in human fibroblast and hepatocyte primary cultures. RKO and Hct116 cells were sensitive to both viruses, whereas LoVo cells were resistant. [(35)S]methionine labeling experiments and viral plaque assays showed that sensitive and resistant colorectal cancer cells supported viral protein and progeny production after infection with either virus. Colorectal cancer cells were pretreated with β-interferon and infected with vesicular stomatitis virus to evaluate the extent to which interferon signaling is downregulated in colorectal cancer cells. Although colorectal cancer cells retained some degree of interferon signaling, this signaling did not negatively impact the oncolytic effects of either virus in sensitive cells. Murine xenografts of RKO cells were effectively treated by intratumoral injections with M protein mutant virus, whereas LoVo xenografts were resistant to treatment with this virus. These results suggest that M protein mutant vesicular stomatitis virus is a good candidate oncolytic virus for the treatment of selected metastatic colorectal cancers.
Virology, 2004
Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attract... more Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.
Susceptibility of breast cancer cells to an oncolytic matrix (M) protein mutant of vesicular stomatitis virus
Cancer Gene Therapy, 2010
Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attrac... more Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attractive candidates as oncolytic viruses for tumor therapies because of their capacity to selectively target cancer cells. The effectiveness of rM51R-M virus as an antitumor agent for the treatment of breast cancer was assessed by determining the ability of rM51R-M virus to infect and kill breast cancer cells in vitro and in vivo. Several human- and mouse-derived breast cancer cell lines were susceptible to infection and killing by rM51R-M virus. Importantly, non-tumorigenic cell lines from normal mammary tissues were also sensitive to VSV infection suggesting that oncogenic transformation does not alter the susceptibility of breast cancer cells to oncolytic VSV. In contrast to results obtained in vitro, rM51R-M virus was only partially effective at inducing regression of primary breast tumors in vivo. Furthermore, we were unable to induce complete regression of the primary and metastatic tumors when tumor-bearing mice were treated with a vector expressing interleukin (IL)-12 or a combination of rM51R-M virus and IL-12. Our results indicate that although breast cancer cells may be susceptible to VSV in vitro, more aggressive treatment combinations are required to effectively treat both local and metastatic breast cancers in vivo.
Journal of Virology, 2003
The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced i... more The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN- promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.
Journal of Virology, 2006
Journal of Virology, 2008
Journal of Virology, 2009
Virology, 1996
The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also a... more The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts of in vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700-11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature. ᭧
Journal of Virology, 2008
Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic virus for tu... more Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic virus for tumor therapies due to its potent tumoricidal activity. Previous studies have demonstrated that VSV selectively infects tumor cells due to defects in their antiviral pathways. These defects make them more susceptible to VSV-induced killing than normal cells. However, some cancer cells display differential sensitivity to VSV. Specifically, LNCaP prostate cancer cells are sensitive to infection with VSV, while PC3 prostate cancer cells are relatively resistant to VSV. This suggests that tumor cells vary in the extent to which they develop defects in antiviral pathways and, thus, permit virus replication. The goal of these studies was to identify the step(s) of the viral replication cycle that is inhibited in PC3 cells. Results showed that although attachment of VSV was not significantly different among cell types, penetration was delayed by 10 to 30 min in PC3 cells relative to LNCaP cells. Primary transcription was delayed by 6 to 8 h in PC3 cells relative to LNCaP cells. Similarly, both secondary transcription and viral protein synthesis rates were delayed by about 6 to 8 h. The progressively increasing delay suggests that more than one step is affected in PC3 cells. Analysis of cellular gene expression showed that in contrast to LNCaP cells, PC3 cells constitutively expressed numerous antiviral gene products, which may enhance their resistance to VSV. These data indicate that the use of VSV for oncolytic virus therapy for prostate tumors may require prescreening of tumors for their level of susceptibility.
Journal of Virology, 2001
During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral gene... more During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral genes is restricted to the latency-associated transcripts (LATs). The stable 2-kb LAT intron has been characterized previously and has been shown to accumulate to high levels in the nuclei of infected neurons. However, in productively infected tissue culture cells, this unique intron is also found in the cytoplasm. Although deletion mutant analysis has suggested that the region of the gene from which the intron is spliced plays a role in maintenance of latency or in reactivation from latency, no well-defined function has been ascribed specifically to the 2-kb LAT intron. Nevertheless, previous work has shown that it associates with 50S particles in the cytoplasm of acutely infected cells. Our studies tested the ability of the 2-kb LAT to dissociate from cytoplasmic protein complexes under various salt conditions. Results indicated that this association, which had been speculated to be mRNA-like, is actually more similar to the affinity of rRNAs for translational complexes. Furthermore, by immunoprecipitation analysis, we demonstrate that the 2-kb LAT associates with ribosomal as well as with splicing complexes in infected cells. Our results suggest that the 2-kb LAT is processed similarly to mRNAs in the nuclei of infected cells. However, in the cytoplasm, the 2-kb LAT may play a structural role in the ribosomal complex, similar to that of the cellular rRNAs, and therefore affect the functioning of the translational machinery.
Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression
Virology, 1997
In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatit... more In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression ...
Vesicular stomatitis virus as a treatment for colorectal cancer
Cancer Gene Therapy, 2011
M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the tr... more M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the treatment of metastatic colorectal cancer due to its ability to kill cancer cells that are defective in their antiviral responses. The oncolytic activity of recombinant wild-type and M protein mutant vesicular stomatitis viruses was determined in RKO, Hct116 and LoVo colorectal cancer cells, as well as in human fibroblast and hepatocyte primary cultures. RKO and Hct116 cells were sensitive to both viruses, whereas LoVo cells were resistant. [(35)S]methionine labeling experiments and viral plaque assays showed that sensitive and resistant colorectal cancer cells supported viral protein and progeny production after infection with either virus. Colorectal cancer cells were pretreated with β-interferon and infected with vesicular stomatitis virus to evaluate the extent to which interferon signaling is downregulated in colorectal cancer cells. Although colorectal cancer cells retained some degree of interferon signaling, this signaling did not negatively impact the oncolytic effects of either virus in sensitive cells. Murine xenografts of RKO cells were effectively treated by intratumoral injections with M protein mutant virus, whereas LoVo xenografts were resistant to treatment with this virus. These results suggest that M protein mutant vesicular stomatitis virus is a good candidate oncolytic virus for the treatment of selected metastatic colorectal cancers.
Virology, 2004
Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attract... more Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.
Susceptibility of breast cancer cells to an oncolytic matrix (M) protein mutant of vesicular stomatitis virus
Cancer Gene Therapy, 2010
Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attrac... more Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attractive candidates as oncolytic viruses for tumor therapies because of their capacity to selectively target cancer cells. The effectiveness of rM51R-M virus as an antitumor agent for the treatment of breast cancer was assessed by determining the ability of rM51R-M virus to infect and kill breast cancer cells in vitro and in vivo. Several human- and mouse-derived breast cancer cell lines were susceptible to infection and killing by rM51R-M virus. Importantly, non-tumorigenic cell lines from normal mammary tissues were also sensitive to VSV infection suggesting that oncogenic transformation does not alter the susceptibility of breast cancer cells to oncolytic VSV. In contrast to results obtained in vitro, rM51R-M virus was only partially effective at inducing regression of primary breast tumors in vivo. Furthermore, we were unable to induce complete regression of the primary and metastatic tumors when tumor-bearing mice were treated with a vector expressing interleukin (IL)-12 or a combination of rM51R-M virus and IL-12. Our results indicate that although breast cancer cells may be susceptible to VSV in vitro, more aggressive treatment combinations are required to effectively treat both local and metastatic breast cancers in vivo.
Journal of Virology, 2003
The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced i... more The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN- promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.
Journal of Virology, 2006
Journal of Virology, 2008
Journal of Virology, 2009
Virology, 1996
The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also a... more The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts of in vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700-11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature. ᭧
Journal of Virology, 2008
Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic virus for tu... more Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic virus for tumor therapies due to its potent tumoricidal activity. Previous studies have demonstrated that VSV selectively infects tumor cells due to defects in their antiviral pathways. These defects make them more susceptible to VSV-induced killing than normal cells. However, some cancer cells display differential sensitivity to VSV. Specifically, LNCaP prostate cancer cells are sensitive to infection with VSV, while PC3 prostate cancer cells are relatively resistant to VSV. This suggests that tumor cells vary in the extent to which they develop defects in antiviral pathways and, thus, permit virus replication. The goal of these studies was to identify the step(s) of the viral replication cycle that is inhibited in PC3 cells. Results showed that although attachment of VSV was not significantly different among cell types, penetration was delayed by 10 to 30 min in PC3 cells relative to LNCaP cells. Primary transcription was delayed by 6 to 8 h in PC3 cells relative to LNCaP cells. Similarly, both secondary transcription and viral protein synthesis rates were delayed by about 6 to 8 h. The progressively increasing delay suggests that more than one step is affected in PC3 cells. Analysis of cellular gene expression showed that in contrast to LNCaP cells, PC3 cells constitutively expressed numerous antiviral gene products, which may enhance their resistance to VSV. These data indicate that the use of VSV for oncolytic virus therapy for prostate tumors may require prescreening of tumors for their level of susceptibility.