Changes in cancer incidence rates by stage during the COVID-19 pandemic in the US.

The coronavirus disease 2019 (COVID-19) pandemic led to health care disruptions and declines in cancer diagnoses in the United States. However, the impact of the pandemic on cancer incidence rates by stage at diagnosis and race and ethnicity is unknown. This cross-sectional study calculated delay- and age-adjusted incidence rates, stratified by stage at diagnosis and race and ethnicity, and rate ratios (RRs) comparing changes in year-over-year incidence rates (eg, 2020 vs 2019) from 2016 to 2020 for 22 cancer types based on data obtained from the Surveillance, Epidemiology, and End Results 22-registry database. From 2019 to 2020, the incidence of local-stage disease statistically significantly declined for 19 of the 22 cancer types, ranging from 4% (RR = 0.96; 95%CI, 0.93-0.98) for urinary bladder cancer to 18% for colorectal (RR = 0.82; 95%CI, 0.81-0.84) and laryngeal (RR = 0.82; 95%CI, 0.78-0.88) cancers, deviating from pre-COVID stable year-over-year changes. Incidence during the corresponding period also declined for 16 cancer types for regional-stage and six cancer types for distant-stage disease. By race and ethnicity, the decline in local-stage incidence for screening-detectable cancers was generally greater in historically marginalized populations. The decline in cancer incidence rates during the first year of the COVID-19 pandemic occurred mainly for local- and regional-stage diseases across racial and ethnic groups. Whether these declines will lead to increases in advanced-stage disease and mortality rates remain to be investigated with additional data years. Nevertheless, the findings reinforce the importance of strengthening the return to preventive care campaigns and outreach for detecting cancers at early and more treatable stages.

Assessment of Changes in Cancer Treatment During the First Year of the COVID-19 Pandemic in the US.

Importance: The COVID-19 pandemic led to disruptions in access to health care, including cancer care. The extent of changes in receipt of cancer treatment is unclear. Objective: To evaluate changes in the absolute number, proportion, and cancer treatment modalities provided to patients with newly diagnosed cancer during 2020, the first year of the pandemic. Design, Setting, and Participants: In this cohort study, adults aged 18 years and older diagnosed with any solid tumor between January 1, 2018, and December 31, 2020, were identified using the National Cancer Database. Data analysis was conducted from September 19, 2022, to July 28, 2023. Exposure: First year of the COVID-19 pandemic. Main Outcomes and Measures: The expected number of procedures for each treatment modality (surgery, radiotherapy, chemotherapy, immunotherapy, and hormonal therapy) in 2020 were calculated using historical data (January 1, 2018, to December 31, 2019) with the vector autoregressive method. The difference between expected and observed numbers was evaluated using a generalized estimating equation under assumptions of the Poisson distribution for count data. Changes in the proportion of different types of cancer treatments initiated in 2020 were evaluated using the additive outlier method. Results: A total of 3 504 342 patients (1 214 918 in 2018, mean [SD] age, 64.6 [13.6] years; 1 235 584 in 2019, mean [SD] age, 64.8 [13.6] years; and 1 053 840 in 2020, mean [SD] age, 64.9 [13.6] years) were included. Compared with expected treatment from previous years' trends, there were approximately 98 000 fewer curative intent surgical procedures performed, 38 800 fewer chemotherapy regimens, 55 500 fewer radiotherapy regimens, 6800 fewer immunotherapy regimens, and 32 000 fewer hormonal therapies initiated in 2020. For most cancer sites and stages evaluated, there was no statistically significant change in the type of cancer treatment provided during the first year of the pandemic, the exception being a statistically significant decrease in the proportion of patients receiving breast-conserving surgery and radiotherapy with a simultaneous statistically significant increase in the proportion of patients undergoing mastectomy for treatment of stage I breast cancer during the first months of the pandemic. Conclusions and Relevance: In this large national cohort study, a significant deficit was noted in the number of cancer treatments provided in the first year of the COVID-19 pandemic. Data indicated that this deficit in the number of cancer treatments provided was associated with decreases in the number of cancer diagnoses, not changes in treatment strategies.

The Burden of Lung Cancer in Women Compared With Men in the US.

This cross-sectional study examines the incidence rates of lung cancer in women compared with men from 2000 to 2019.

Changes in cancer diagnoses and stage distribution during the first year of the COVID-19 pandemic in the USA: a cross-sectional nationwide assessment.

BACKGROUND: The emergence of COVID-19 disrupted health care, with consequences for cancer diagnoses and outcomes, especially for early stage diagnoses, which generally have favourable prognoses. We aimed to examine nationwide changes in adult cancer diagnoses and stage distribution during the first year of the COVID-19 pandemic by cancer type and key sociodemographic factors in the USA. METHODS: In this cross-sectional study, adults (aged ≥18 years) newly diagnosed with a first primary malignant cancer between Jan 1, 2018, and Dec 31, 2020, were identified from the US National Cancer Database. We included individuals across 50 US states and the District of Columbia who were treated in hospitals that were Commission on Cancer-accredited during the study period. Individuals whose cancer stage was 0 (except for bladder cancer), occult, or without an applicable American Joint Committee on Cancer staging scheme were excluded. Our primary outcomes were the change in the number and the change in the stage distribution of new cancer diagnoses between 2019 (Jan 1 to Dec 31) and 2020 (Jan 1 to Dec 31). Monthly counts and stage distributions were calculated for all cancers combined and for major cancer types. We also calculated annual change in stage distribution from 2019 to 2020 and adjusted odds ratios (aORs) using multivariable logistic regression, adjusted for age group, sex, race and ethnicity, health insurance status, comorbidity score, US state, zip code-level social deprivation index, and county-level age-adjusted COVID-19 mortality in 2020. Separate models were stratified by sociodemographic and clinical factors. FINDINGS: We identified 2 404 050 adults who were newly diagnosed with cancer during the study period (830 528 in 2018, 849 290 in 2019, and 724 232 in 2020). Mean age was 63·5 years (SD 13·5) and 1 287 049 (53·5%) individuals were women, 1 117 001 (46·5%) were men, and 1 814 082 (75·5%) were non-Hispanic White. The monthly number of new cancer diagnoses (all stages) decreased substantially after the start of the COVID-19 pandemic in March, 2020, although monthly counts returned to near pre-pandemic levels by the end of 2020. The decrease in diagnoses was largest for stage I disease, leading to lower odds of being diagnosed with stage I disease in 2020 than in 2019 (aOR 0·946 [95% CI 0·939-0·952] for stage I vs stage II-IV); whereas, the odds of being diagnosed with stage IV disease were higher in 2020 than in 2019 (1·074 [1·066-1·083] for stage IV vs stage I-III). This pattern was observed in most cancer types and sociodemographic groups, although was most prominent among Hispanic individuals (0·922 [0·899-0·946] for stage I; 1·110 [1·077-1·144] for stage IV), Asian American and Pacific Islander individuals (0·924 [0·892-0·956] for stage I; 1·096 [1·052-1·142] for stage IV), uninsured individuals (0·917 [0·875-0·961] for stage I; 1·102 [1·055-1·152] for stage IV), Medicare-insured adults younger than 65 years (0·909 [0·882-0·937] for stage I; 1·105 [1·068-1·144] for stage IV), and individuals living in the most socioeconomically deprived areas (0·931 [0·917-0·946] for stage I; 1·106 [1·087-1·125] for stage IV). INTERPRETATION: Substantial cancer underdiagnosis and decreases in the proportion of early stage diagnoses occurred during 2020 in the USA, particularly among medically underserved individuals. Monitoring the long-term effects of the pandemic on morbidity, survival, and mortality is warranted. FUNDING: None.

Disparities and Trends in Genitourinary Cancer Incidence and Mortality in the USA.

BACKGROUND: Previous studies have reported on incidence and mortality patterns for individual genitourinary cancers in the USA. However, these studies addressed individual cancer types rather than genitourinary cancers overall. OBJECTIVE: To comprehensively examine disparities and trends in the incidence and mortality for the four major genitourinary cancers (bladder, kidney, prostate, and testis) in the USA. DESIGN, SETTING, AND PARTICIPANTS: We obtained incidence data from the National Cancer Institute 22-registry Surveillance, Epidemiology and End Results (SEER) database and the US Cancer Statistics database (Centers for Disease Control and Prevention) and mortality data from the National Center for Health Statistics to examine cross-sectional and temporal trends in incidence and death rates stratified by sex, race/ethnicity, and county. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Age-adjusted incidence and death rates were calculated using SEER*Stat software. Temporal trends were analyzed using Joinpoint regression for a two-sided significance level of p < 0.05. RESULTS AND LIMITATIONS: Incidence and mortality rates for bladder and kidney cancers were two to four times higher for men than for women. Among non-Hispanic White individuals, the highest incidence rates were found in the Northeast for bladder cancer and in Appalachia for kidney cancer, whereas the highest death rates for prostate cancer were found in the West. Incidence rates increased for cancers of the kidney and testis and for advanced-stage prostate cancer in almost all racial/ethnic populations and for bladder cancer in the American Indian/Alaska Native population. Death rates increased for testicular cancer in the Hispanic population and stabilized for prostate cancer among White and Asian American/Pacific Islander men after a steady decline since the early 1990s. Study limitations include misclassification of race/ethnicity on medical records and death certificates. CONCLUSIONS: We found persistent sociodemographic disparities and unfavorable trends in incidence or mortality for all four major genitourinary cancers. Future studies should elucidate the reasons for these patterns. PATIENT SUMMARY: In the USA, rates of cancer cases are increasing for kidney, testis, and advanced-stage prostate cancers in the overall population, and for bladder cancer in the American Indian/Alaska Native population. Differences in the rates by sex and race/ethnicity remain.

Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity.

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Foot-and-mouth disease virus 5'-terminal S fragment is required for replication and modulation of the innate immune response in host cells.

The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.

Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity.

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.

Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

BACKGROUND: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3C(pro), and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. METHODS: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. RESULTS: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3D(pol) and 3C(pro) consistent with early observations of FMDV 3C(pro)-induced cleavage of Sam68. CONCLUSION: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C(pro) and 3D(pol) and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3C(pro) and 3D(pol).

Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of foot-and-mouth disease virus in host cells.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus (FMDV) that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergency response measures such as therapeutic treatments, are on high demand. In this study, we analyzed the long-term exposure outcome to a previously identified inhibitor of 3D polymerase (FMDV 3Dpol) for controlling FMDV infection and for the selection of resistance mutants. The results showed that no escape mutant viruses were isolated from FMDV A24 Cruzeiro infections in cell culture treated with gradually increasing concentrations of the antiviral compound 5D9 (4-chloro-N'-thieno, [2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide) over ten passages. Biochemical and plaque assays revealed that when 5D9 was used at concentrations within a non-toxic range in cells, it drove the virus to undetectable levels at passage eight to ten. This is in contrast with observations made on parallel control (untreated) passages exhibiting fully viable and stable virus progenies. Collectively, the results demonstrated that under the experimental conditions, treatment with 5D9 does not confer a resistant phenotype and the virus is unable to evade the antiviral effect of the inhibitor. Further efforts using quantitative structure-property relationship (QSPR) based modifications of the 5D9 compound may result in the successful development of an effective in vivo antiviral drug targeting FMDV.

The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells.

Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C(pro) induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5' non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C(pro).

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase.

BACKGROUND: Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. METHODOLOGY/PRINCIPAL FINDINGS: Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC(50)s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC(50) = 12 µM and EC(90) = 20 µM). SIGNIFICANCE: We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.

Dendritic cells infected with vpr-positive human immunodeficiency virus type 1 induce CD8+ T-cell apoptosis via upregulation of tumor necrosis factor alpha.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.

Antiviral effects of mifepristone on human immunodeficiency virus type-1 (HIV-1): targeting Vpr and its cellular partner, the glucocorticoid receptor (GR).

The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the optimal infection of primary monocytes/macrophages and other non-dividing cells. Vpr interacts with the cellular glucocorticoid receptor (GR) and transactivates the HIV-1 LTR through glucocorticoid response element (GRE), an event that can be blocked by the GR antagonist, mifepristone. Results demonstrated that Vpr-induced transactivation of the HIV-1 LTR was inhibited by mifepristone in a dose-dependent manner by >60% at a 10 microM concentration. Infectivity assays using X4 and R5 viruses demonstrated antiviral effects on a dose-dependent regimen of mifepristone. The effects of mifepristone were also tested in latently infected cells that could be activated with extracellular Vpr protein and results indicated specific inhibition of virus reactivation in the presence of this antagonist.

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis.

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).

Human immunodeficiency virus type 1 Vpr impairs dendritic cell maturation and T-cell activation: implications for viral immune escape.

Antigen presentation and T-cell activation are dynamic processes involving signaling molecules present in both APCs and T cells. Effective APC function and T-cell activation can be compromised by viral immune evasion strategies, including those of human immunodeficiency virus type 1 (HIV-1). In this study, we determined the effects of HIV-1 Vpr on one of the initial target of the virus, dendritic cells (DC), by investigating DC maturation, cytokine profiling, and CD8-specific T-cell stimulation function followed by a second signal. Vpr impaired the expression of CD80, CD83, and CD86 at the transcriptional level without altering normal cellular transcription. Cytokine profiling indicated that the presence of Vpr inhibited production of interleukin 12 (IL-12) and upregulated IL-10, whereas IL-6 and IL-1beta were unaltered. Furthermore, DC infected with HIV-1 vpr+ significantly reduced the activation of antigen-specific memory and recall cytotoxic-T-lymphocyte responses. Taken together, these results indicate that HIV-1 Vpr may in part be responsible for HIV-1 immune evasion by inhibiting the maturation of costimulatory molecules and cytokines essential for immune activation.

Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication.

HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL, translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr-GR interaction, subcellular localization and its effect on Vpr-GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher level of activation compared to Vprwt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift assay (EMSA) revealed that the VprL22A-GR complex had higher DNA-binding activity when compared to the wild type Vpr-GR complex. These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.

Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus.

HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.