Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.     

Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.     

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.     

The multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle

The Vif (virion infectivity factor protein of human immunodeficiency virus type I (HIV-1) is essential for viral replication in vivo and productive infection of peripheral blood mononuclear cells, macrophages, and H9 T-cells. However, the molecular mechanism(s) of Vif remains unknown and needs to be further determined. In this report, we show that, like many other proteins encoded by HIV-1, Vif proteins possess a strong tendency toward self-association. In relatively native conditions, Vif proteins formed multimers in vitro, including dimers, trimers, or tetramers. Through in vivo binding assays such as coimmunoprecipitation and the mammalian two-hybrid system, we also demonstrated that Vif proteins could interact with each other within a cell, indicating that the multimerization of Vif proteins is not simply due to fortuitous aggregation. Further studies indicated that the domain affecting Vif self-association is located at the C terminus of this protein, especially the proline-enriched 151-164 region. Moreover, we found that a Vif mutant with deletion at amino acid 151-164 was unable to rescue the infectivity of vif-defective viruses generated from H9 T-cells, suggesting that the multimerization of Vif proteins could be important for Vif function in the viral life cycle. Our studies identified a new feature of Vif and should accelerate our understanding of its role in HIV-1 pathogenesis.     

Infection of a yellow baboon with simian immunodeficiency virus from African green monkeys: evidence for cross-species transmission in the wild.

Many African primates are known to be naturally infected with simian immunodeficiency viruses (SIVs), but only a fraction of these viruses has been molecularly characterized. One primate species for which only serological evidence of SIV infection has been reported is the yellow baboon (Papio hamadryas cynocephalus). Two wild-living baboons with strong SIVAGM seroreactivity were previously identified in a Tanzanian national park where baboons and African green monkeys shared the same habitat (T. Kodama, D. P. Silva, M. D. Daniel, J. E. Phillips-Conroy, C. J. Jolly, J. Rogers, and R. C. Desrosiers, AIDS Res. Hum. Retroviruses 5:337-343, 1989). To determine the genetic identity of the viruses infecting these animals, we used PCR to examine SIV sequences directly in uncultured leukocyte DNA. Targeting two different, nonoverlapping genomic regions, we amplified and sequenced a 673-bp gag gene fragment and a 908-bp env gene fragment from one of the two baboons. Phylo-genetic analyses revealed that this baboon was infected with an SIVAGM strain of the vervet subtype. These results provide the first direct evidence for simian-to-simian cross-species transmission of SIV in the wild.     

Bean yellow disorder virus: Parameters of transmission by Bemisia tabaci and host plant range

Abstract Bean yellow disorder virus (BnYDV) was recently identified as the first crinivirus (family Closteroviridae) that infects members of the family Leguminosae. It was first observed during the autumn of 2003, causing heavy losses in French bean (Phaseolus vulgaris L.) grown commercially in Spain. The virus is transmitted by the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) Q‐biotype, and disease symptoms resemble nutritional disorders consisting of interveinal mottling and yellowing in leaves, combined with stiffness or brittleness, and are typically produced on the middle to lower parts of the plant. Transmission experiments showed that 50% and 100% of B. tabaci adults acquired the virus after a feeding period of 3 and 7 h, respectively. Viruliferous whiteflies infected 66% and 100% of P. vulgaris plants after a feeding period of 12 and 24 h, respectively. The transmission efficiency of single whiteflies was 37% and persistence of BnYDV in the vector lasted up to 2 weeks with a half‐life of 9 days. BnYDV was transmitted to P. vulgaris, Pisum sativum L., Lens culinaris Medik., and Vicia faba L., but not to Vigna unguiculata L., Glycine max (L.) Merr., Cicer arietum L., and to crop species belonging to families of the Solanaceae and Cucurbitaceae. No virus was detected in field samples collected from 30 different species from Boraginaceae, Asteraceae, Geraniaceae, Lamiaceae, Leguminosae, Malvaceae, Scrophulariaceae, Thymelaeaceae and Verbenaceae. The restricted host range and efficient management of crops regarding whitefly infestation may be key elements in the control of BnYDV.     

Amino acid residues 88 and 89 in the central hydrophilic region of human immunodeficiency virus type 1 Vif are critical for viral infectivity by enhancing the steady-state expression of Vif

A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E(88)WRKKR(93)) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E(88) or W(89) residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E(88) and W(89) with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E(88) or W(89); a mutant with substitution of E(88) and W(89) with A; and a mutant with substitution of E(88), W(89), and R(90) with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E(88) and W(89) residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.     

Deletions in the transmembrane domain of a sindbis virus glycoprotein alter virus infectivity, stability, and host range

The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and protein-membrane interactions during virus assembly.     

Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant.

Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.     

Evidence that the structural conformation of envelope gp120 affects human immunodeficiency virus type 1 infectivity, host range, and syncytium-forming ability.

We investigated how amino acid changes within and outside the V3 loop of the envelope glycoprotein of human immunodeficiency virus type 1 influence the infectivity, host range, and syncytium-forming ability of the virus. Our studies show that on the genomic backgrounds of the human immunodeficiency virus type 1 strains SF2 and SF13, a reciprocal exchange of full-loop sequences does not alter the syncytium-forming ability of the viruses, indicating that a determinant(s) for this biological property maps outside the loop. However, specific amino acid substitutions, both within and outside the V3 loop, resulted in loss of infectivity, host range, and syncytium-forming potential of the virus. Furthermore, it appears that a functional interaction of the V3 loop with regions in the C2 domain of envelope gp120 plays a role in determining these biological properties. Structural studies of mutant glycoproteins show that the mutations introduced affect the proper association of gp120 with the transmembrane glycoprotein gp41. Our results suggest that mutations that alter the structure of the V3 loop can affect the overall conformation of gp120 and that, reciprocally, the structure of the V3 loop is influenced by the conformation of other regions of gp120. Since the changes in the replicative potential, host range, and fusogenic ability of the mutant viruses correlate well with the changes in gp120 conformation, as monitored by the association of gp120 with gp41, our results support a close relationship between envelope gp120 structural conformation and the biological phenotype of the virus.     

Aphid transmission of cauliflower mosaic virus: The role of the host plant

Transmission of plant viruses is the result of interactions between a given virus, the host plant and the vector. Most research has focused on molecular and cellular virus-vector interactions, and the host has only been regarded as a reservoir from which the virus is acquired by the vector more or less accidentally. However, a growing body of evidence suggests that the host can play a crucial role in transmission. Indeed, at least one virus, Cauliflower mosaic virus, exploits the host''s cellular pathways to form specialized intracellular structures that optimize virus uptake by the vector and hence transmission.Key words: virus, vector, host plant, transmission, interactionsTransmission is a step in a virus''s life cycle that is often neglected. Nonetheless, it is obvious that also this step is obligatory for a virus, as it could not maintain itself without dispersing to other hosts and infecting them. Most plant viruses are transmitted by insects, using two different strategies: “circulant transmission” where the virus, once taken up by the vector during feeding on an infected plant, passes from the intestine via the body lumen to the salivary glands and is finally inoculated with the saliva into a new host plant; the second strategy is “non-circulant transmission” where transmissible virus particles attach only to the exterior mouthpieces of the insect from which they are released into a new host. Whereas the first strategy obviously requires highly specific interactions between the virus and the vector to allow for passage of the virus through the vector, non-circulant transmission was initially thought of as a more or less accidental event, where virus sticks non-specifically to the mouthpieces. However, it becomes more and more evident that also non-circulant transmission is the result of sophisticated interactions between a given virus, a host and a vector. The vectors are most often aphids that, due to their non-destructive feeding behavior, are ideally suited as virus vectors. In fact, once landed on a plant, aphids first probe the prospective food source by short, only seconds lasting intracellular punctures in epidermis and mesophyll cells that do not even kill the punctured cells.¹ After these exploratory punctures and when they judge the plant as suited, the aphids insert their proboscis-like mouthpieces (stylets) into the phloem and feed from its sap for time spans that may exceed several hours. Depending on the tissues they infect, plant viruses can be acquired by aphids during either or only one of the two puncture phases. For example, Luteoviruses are only acquired from the vascular tissues,² whereas Cauliflower mosaic virus is acquired from both tissues.³Cauliflower mosaic virus (CaMV) is one of the best studied viruses on what concerns non-circulant transmission, the most often used transmission mode employed by plant viruses. For its transmission, a transmissible complex must form that attaches to a protein receptor located in the stylets of the aphid.⁴ This complex is not only, as for some viruses, composed of the virus particle, but also, as for many non-circulantly transmitted plant viruses, of a viral helper protein that with one domain interacts with the virus particle and with another with the stylet receptor⁵ (Fig. 1A). The helper protein of CaMV, P2, seems to have no other function but to assist in transmission as CaMV mutants deleted of P2 are perfectly infectious but not transmissible.⁶ A puzzling fact is that P2 may be acquired independently of the virus particle, meaning that it alone can bind to the stylet receptor and that virus particles either attach concomitantly with P2 onto the stylets or later attach to pre-bound P2. This has consequences for the composition of the transmitted viral population as it can be compiled of virus particles originating from the same cell from which P2 was acquired, but also from other cells and even sieve tubes that themselves do not contain P2.³ In fact, this potentially sequential acquisition mode of CaMV by the vector is controlled by the intracellular⁷ and tissue-specific localization of P2 that is only found in epidermis and parenchyma cells.³ In these cells, P2 localizes exclusively in a single viral inclusion, the transmission body, that has been proposed and recently been shown to be specialized for transmission:^8–10 if this structure does not form, CaMV can not be taken up by the aphid, even if functional P2 is present in the infected cell.Open in a separate windowFigure 1(A) The different strategies of non-circulant transmission: Viruses (V) using the capsid strategy (CS) attach directly to a receptor (R) in the tip of the a proboscis forming aphid stylets (blue), whereas in the helper strategy (HS) this interaction is mediated by the viral helper protein (H) that binds the virus particle to the receptor. Note that the helper protein can bind independently of the virus to the stylets. Whether the same receptor is used by different viruses as presented in the schema, is not known. (B) A turnip protoplast transfected with CaMV was double-labelled late in infection for CaMV helper protein P2 (red) and the marker protein for the virus factories P6 (green). It is visible that P2 localizes in a single, large transmission body, whereas the numerous virus factories are devoid of P2 (Colocalization would be revealed by yellowish color (M) in this superposition). (C and D) Turnip protoplasts were cotransfected with CaMV and TBK5-GFP and immunolabelled for P2 (red) and TBK5 was detected by GFP fluorescence. (C) shows a cell early in infection, where P2 and TBK5-GFP colocalize on a network that we identified as the microtubule cytoskeleton (unpublished data). (D) shows a cell later in infection where P2 and TBK5-GFP colocalize, as indicated by the yellowish color, in a transmission body. Note that TBK5-GFP also strongly labels the nucleus (N).This posed the interesting question how the transmission body forms during infection because elucidating this mechanism would show that CaMV hijacks cellular pathways for the sole purpose to ensure its transmission. It was known that besides the single transmission body a second type of viral inclusion bodies is found in infected cells: the numerous “electron-dense inclusions” that are assumed to be the virus factories (Fig. 1B) where all viral synthesis occurs¹¹ and where most virus particles accumulate. However, P2 was never described in the factories, presenting the paradox: if it is translated in the factories why is it not found there? Of different possible scenarios we chose to test the hypothesis that P2 is produced in the factories and then exported. Protoplasts were transfected with CaMV particles and kinetics of P2 accumulation followed by immunofluorescence. The results showed that P2 is indeed translated in the viral factories but then associates temporally with microtubules before finally condensing into a single transmission body. Also the other known components of the transmission body, the viral protein P3 and to a lesser degree, some virus particles, followed the same route from viral factories to the transmission body.Experiments with cytoskeleton drugs confirmed that transient localization of transmission body components with microtubules, but not with actin filaments, is necessary for transmission body formation. The results also indicated that both microtubules and actin filaments are apparently not required for other steps of the intracellular infection cycle because formation of viral factories was only slightly inhibited by the drugs.The results show that CaMV specifically uses the microtubule cytoskeleton to form the transmission body and thus enable vector transmission. Consequently, non-circulant transmission of at least this virus is not a random event where the vector takes up some transmissible complexes by chance. It is rather the result of highly specific interactions, where the virus “intentionally” (ab)uses cellular pathways to optimize acquisition by the vector, and this long before arrival of the latter on an infected plant.A lot of questions remain open, though. Are P2 and the other components of the transmission body actively transported on microtubules, or is their transient colocalization with microtubules part of an alternative transport mode? We started to more closely examine interaction between P2 and microtubules and privileged the hypothesis that the protein might be transported by a motor activity on microtubules. As preliminary data indicated that P2 does not possess an innate translocating activity, we looked for a cellular motor protein and tested as a candidate the kinesin TBK5.¹² This transport protein is, when overexpressed, able to bundle microtubules into a single focus, just as transmission bodies are singular structures in the cell. When healthy protoplasts were cotransfected with TBK5 and CaMV, TBK5 localized transiently with P2 on microtubules and in transmission bodies (Fig. 1C and D). This might be taken as the first evidence that a kinesin might be involved in formation of transmission bodies, but more experimentation is needed to confirm this hypothesis.A by far more important question is: Have also other viruses, whether from the plant or the animal kingdoms, that are noncirculantly (or mechanically, as animal virologists call this mode of transmission) transmitted, developed similar strategies that fine-tune interactions between the host and the virus to prepare and perfect transmission?     

Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity.

Accurate and sensitive quantitation of infectious human immunodeficiency virus (HIV) has been difficult to achieve. In this report, a quantitative focal immunoassay (FIA) for HIV was developed using human HeLa cells rendered susceptible to HIV infection by introduction of the CD4 gene via a retrovirus vector. Infected cells were identified by using human anti-HIV antibodies or mouse monoclonal antibodies specific for HIV together with secondary fluorescein- or peroxidase-conjugated antibody specific for mouse or human immunoglobulins. The assay identified cells infected with either wild-type or culture-adapted HIV isolates and was capable of detecting 1 positive cell in 10(6) cells. The FIA was also effective at detecting cell-free HIV, and in contrast to assays using A3.01, CEM, and other human leukemia cells, the FIA detected most wild-type HIV isolates. HIV neutralization could be determined by using the FIA, and two monoclonal antibodies reactive with HIV gp120 were found to neutralize only the LAV-IIIB strain of HIV. These monoclonal antibodies, as well as antibodies in serum samples from patients with acquired immune deficiency syndrome, were able to inhibit the spread of HIV infection in human lymphocyte suspension cultures but not in CD4-positive HeLa cells growing attached to plastic dishes.     

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

Genetic differences accounting for evolution and pathogenicity of simian immunodeficiency virus from a sooty mangabey monkey after cross-species transmission to a pig-tailed macaque.

We determined the nucleotide sequences of two related isolates of simian immunodeficiency virus from the sooty mangabey monkey (SIVsmm) that exhibit dramatic differences in virulence. These isolates are separated by one experimental cross-species transmission, from sooty mangabey to pig-tailed macaque. The parental virus (SIVsmm9), nonpathogenic in the original host (sooty mangabeys), causes a chronic AIDS-like disease in macaques. In contrast, the variant virus (SIVsmmPBj14) induces an acute lethal disease in various macaque species and is also pathogenic for sooty mangabeys. The combination of necessary and sufficient mutations that determined the acutely lethal phenotype on the SIVsmm9 genetic background is included within a maximal set of 57 point mutations, plus two insertions located in the long terminal repeat (22 bp spanning an NF-kappa B-like enhancer element) and in the surface envelope glycoprotein (5 amino acids). Comparisons of synonymous and nonsynonymous nucleotide substitutions in the genome of SIVsmm indicated that selective pressures, probably due to the host immune response, favored amino acid changes in the envelope. This immunoevolutionary mechanism could explain the increase in diversity and the apparition of new virulent phenotypes after cross-species transmission.     

Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and Virion encapsidation

The human immunodeficiency virus type-1 (HIV-1) accessory protein Vif serves to neutralize the human antiviral proteins apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G [A3G]) and A3F. As such, the therapeutic blockade of Vif function represents a logical objective for rational drug design. To facilitate such endeavors, we have employed molecular genetics to define features of A3G that are required for its interaction with Vif. Using alanine-scanning mutations and multiple different substitutions at key residues, we confirm the central role played by the aspartic acid at position 128 and identify proline 129 and aspartic acid 130 as important contributory residues. The overall negative charge of this 3-amino-acid motif appears critical for recognition by Vif, as single lysine substitutions are particularly deleterious and a double alanine substitution at positions 128 and 130 is far more inhibitory than single-residue mutations at either position. Our analyses also reveal that the immediately adjacent 4 amino acids, residues 124 to 127, are important for the packaging of A3G into HIV-1 particles. Most important are tyrosine 124 and tryptophan 127, and mutations at these positions can ablate virion incorporation, as well as the capacity to inhibit virus infection. Thus, while pharmacologic agents that target the acidic motif at residues 128 to 130 have the potential to rescue A3G expression by occluding recognition by Vif, care will have to be taken not to perturb the contributions of the neighboring 124-to-127 region to packaging if such agents are to have therapeutic benefit by promoting A3G incorporation into progeny virions.