Functional analysis of nuclear localization signals in VP1-2 homologues from all herpesvirus subfamilies.

UNLABELLED: The herpes simplex virus (HSV) tegument protein VP1-2 contains an N-terminal nuclear localization signal (NLS) that is critical for capsid routing to the nuclear pore. Here we analyzed positionally conserved determinants in VP1-2 homologues from each of the alpha, beta, and gamma classes of human herpesviruses. The overall architectures of the VP1-2s were similar, with a conserved N-terminal ubiquitin-specific protease domain separated from an internal region by a linker that was quite poorly conserved in length and sequence. Within this linker region all herpesviruses contained a conserved, highly basic motif which nevertheless exhibited distinct class-specific features. The motif in HSV functioned as a monopartite NLS, while in varicella-zoster virus (VZV) activity required an adjacent basic section defining the motif as a bipartite NLS. Neither the beta- nor gammaherpesvirus VP1-2 motifs were identified by prediction algorithms, but they nevertheless functioned as efficient NLS motifs both in heterologous transfer assays and in HSV VP1-2. Furthermore, though with different efficiencies and with the exception of human herpesvirus 8 (HHV-8), these chimeric variants rescued the replication defect of an HSV mutant lacking its NLS motif. We demonstrate that the lysine at position 428 of HSV is critical for replication, with a single alanine substitution being sufficient to abrogate NLS function and virus growth. We conclude that the basic motifs of each of the VP1-2 proteins are likely to confer a similar function in capsid entry in the homologous setting and that while there is flexibility in the exact type of motif employed, specific individual residues are critical for function. IMPORTANCE: To successfully infect cells, all herpesviruses, along with many other viruses, e.g., HIV, hepatitis B virus, and influenza virus, must navigate through the cytoplasmic environment and dock with nuclear pores for transport of their genomes into the nucleus. However, we still have a limited understanding of the detailed mechanisms involved. Insight into these events is needed and could offer opportunities for therapeutic intervention. This work investigated the role of a specific determinant in the structural protein VP1-2 in herpesvirus entry. We examined this determinant in representative VP1-2s from all herpesvirus subfamilies, demonstrated NLS function, dissected key residues, and showed functional relevance in rescuing replication of the mutant blocked in capsid navigation to the pore. The results are important and strongly support our conclusions of the generality that these motifs are crucial for entry of all herpesviruses. They also facilitate future analysis on selective host interactions and possible routes to disrupt function.

A Nuclear localization signal in herpesvirus protein VP1-2 is essential for infection via capsid routing to the nuclear pore.

To initiate infection, herpesviruses must navigate to and transport their genomes across the nuclear pore. VP1-2 is a large structural protein of the virion that is conserved in all herpesviruses and plays multiple essential roles in virus replication, including roles in early entry. VP1-2 contains an N-terminal basic motif which functions as an efficient nuclear localization signal (NLS). In this study, we constructed a mutant HSV strain, K.VP1-2ΔNLS, which contains a 7-residue deletion of the core NLS at position 475. This mutant fails to spread in normal cells but can be propagated in complementing cell lines. Electron microscopy (EM) analysis of infection in noncomplementing cells demonstrated capsid assembly, cytoplasmic envelopment, and the formation of extracellular enveloped virions. Furthermore, extracellular virions isolated from noncomplementing cells had similar profiles and abundances of structural proteins. Virions containing VP1-2ΔNLS were able to enter and be transported within cells. However, further progress of infection was prevented, with at least a 500- to 1,000-fold reduction in the efficiency of initiating gene expression compared to that in the revertant. Ultrastructural and immunofluorescence analyses revealed that the K.VP1-2ΔNLS mutant was blocked at the microtubule organizing center or immediately upstream of nuclear pore docking and prior to gene expression. These results indicate that the VP1-2 NLS is not required for the known assembly functions of the protein but is a key requirement for the early routing to the nuclear pore that is necessary for successful infection. Given its conservation, we propose that this motif may also be critical for entry of other classes of herpesviruses.

Autocatalytic activity of the ubiquitin-specific protease domain of herpes simplex virus 1 VP1-2.

The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances.

A single mutation responsible for temperature-sensitive entry and assembly defects in the VP1-2 protein of herpes simplex virus.

Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.

Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7.

VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein.

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

Enhanced CD8+ T cell immune response against a V3 loop multi-epitope polypeptide (TAB13) of HIV-1 Env after priming with purified fusion protein and booster with modified vaccinia virus Ankara (MVA-TAB) recombinant: a comparison of humoral and cellular immune responses with the vaccinia virus Western Reserve (WR) vector.

The humoral and cytotoxic T-lymphocyte (CTL) responses have been shown to be determinant in the clearance of many viral infections and because of those characteristics, vaccine candidates against AIDS are designed to enhance both arms of the immune system. While a protocol of immunization able to confer protection in humans against HIV will have to await the results of current clinical trials, it remains important to identify protocols of immunization in animals that achieve significant levels of humoral and cellular immune responses to HIV. In this study we have carried out a comparative analysis of the immune responses elicited in mice immunized with recombinants based on the modified vaccinia virus Ankara strain (rMVA) versus the Western Reserve strain (WR) of vaccinia virus (rVV), both expressing a V3 loop multi-epitopic protein from eight different HIV isolates (TAB13). We found that during priming, rMVA elicited a two- to three-fold higher specific CD8+ T cell response than rVV. Similar enhancement was observed during priming with purified protein TAB13 followed by a booster with rMVA. The epitopes LR150, MN and IIIB, located at the ends and in the middle of the chimeric protein, were able to induce a specific CD8+ T cell response, both after priming or prime/booster with the recombinant viruses but not after prime/booster with TAB13. By examining the cytokine pattern, the immune response triggered by these vectors was of Th-1 type. Humoral immune responses were higher in animals immunized with TAB13/TAB13 or TAB13/rVV than in animals immunized with TAB13/rMVA. These findings demonstrate that during priming or in a prime/booster immunizations, rMVA is superior to rVV in the ability to enhance specific cellular responses to an HIV-1 protein, and that both humoral and cellular immune responses to theV3 loop epitope of HIV-1 Env can be obtained by priming with TAB13 followed by a booster with viral vectors.

[Distribution of lipidic values in teenagers that smoke]. / Distribución de las variables lipídicas en ádolescentes fumadores.

OBJECTIVE: To determine the lipidic profiles in a population of teenagers and determine their relationship with smoking. PATIENTS AND METHODS: We have studied 910 teenagers, between 14 and 17 years of age. Information regarding smoking was obtained with a questionnaire. Serum levels of cholesterol, HDL, LDL, triglycerides, apoprotein A and apoprotein B were measured by enzymatic methods. RESULTS: Total cholesterol of non-smokers is higher than in smokers. Even if we divide the population studied by gender, the difference remain. Nevertheless, smokers' mean HDL level is lower, especially in boys. The LDL/HDL index is slightly higher in smokers than in non-smokers. However, if we separate boys and girls, we found some differences. Boys that smoke have higher cholesterol/HDL and LDL/HDL than non-smoking boys. Adjusting lipidic values by body mass, age and triglyceride serum levels, the relationship between smoking and lower cholesterol, HDL and apoprotein A and higher serum triglyceride levels remains in boys.

Resistance to candidiasis and macrophage activity in chitin-treated mice.

The effect of chitin, a polysaccharide of the cell wall of Candida albicans, on both the survival of C. albicans infected mice and the activity of the murine peritoneal macrophages has been studied. Pretreatment of mice with 30 mg kg(-1) C. albicans chitin enhanced the survival of the infected animals. The protective effect was concomitant with an enhancement of both phagocytic and candidacidal activities of the peritoneal macrophages. Chitin by itself did not induce the nitric oxide (NO) synthase in the macrophages, which remained at a level similar to that shown by the macrophages from untreated animals. The administration of 10 mg kg(-1) C. albicans chitin diminished the long term survival of the infected animals. This effect was coincident with a lower candidacidal activity and NO production by the macrophages of the chitin treated and infected animals, compared to the untreated infected animals.

[Changes in mean serum lipid levels in pediatric and adolescent population of Navarra between 1987 and 1993]. / Modificaciones en los niveles medios de lípidos séricos en la población infantil y juvenil de Navarra entre 1987 y 1993.

INTRODUCTION AND OBJECTIVES: Atherosclerosis originates during childhood and serum lipid levels are a key factor of the process. The aim of this study is to assess the evolution in the mean serum lipid levels found in children and teenagers in Navarra, Spain, in 1987 and 1993 and to compare them with the overall Spanish standard levels. METHODS: Two cross-sectional surveys on cardiovascular risk factors in the population aged 4 to 17-years in the Navarra region, carried out in 1987 and 1993. Both studies used comparable procedures, and covered school-children between the above-mentioned ages of 4 and 17 years. The lipid levels were assayed by enzymatic methods from blood samples, using an autoanalyzer. Statistical methods to compare means and proportions were used. RESULTS: The sample size was 5,829 in 1987 and 3,256 in 1993. Among males, the average cholesterolemia was 177.3 mg/dl in 1987 and 174 mg/dl in 1993. Among females it was 179.8 mg/dl in 1987 and 174.9 mg/dl in 1993. The percentage of males with cholesterolemia over 200 mg/dl was 20.4% in 1987 and 16.8% in 1993. Among females this percentage was 21.9% in 1987 and 17.8% in 1993. The percentage of males showing hyperlipidemia (LDL/HDL > 2.2) was 15.1% in 1987 and 12.5% in 1993. For females this percentage went from 16.8% in 1987 to 13% in 1993. CONCLUSIONS: Between 1987 and 1993 a decrease in the average levels of serum cholesterol and LDL-cholesterol among the infant-to-young population of Navarra has been assessed. No changes in the HDL or serum tryglicerides were detected. Some effects in the results, due to differences in the pre-analytical and analytical phases of both surveys, cannot be excluded.