Two different molecular defects in the Tva receptor gene explain the resistance of two tvar lines of chickens to infection by subgroup A avian sarcoma and leukosis viruses

The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tva(s) encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tva(s) cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tva(s) cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 7(2). Line C tva(r) contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of Tva(R) for the ASLV(A) envelope glycoproteins. Line 7(2) tva(r2) contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.     

The receptor for the subgroup C avian sarcoma and leukosis viruses, Tvc, is related to mammalian butyrophilins, members of the immunoglobulin superfamily

The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.     

A single-amino-acid substitution in the TvbS1 receptor results in decreased susceptibility to infection by avian sarcoma and leukosis virus subgroups B and D and resistance to infection by subgroup E in vitro and in vivo

The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type Tvb^S1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvb^r2, encodes a mutant Tvb^S1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in Tvb^S1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the Tvb^S1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.     

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.     

Selection of an avian retrovirus mutant with extended receptor usage.

Receptor recognition by avian retroviruses is thought to involve the interaction of two regions of the SU protein, hr1 and hr2, with the host cell surface receptor. These regions exhibit considerable variation, concordant with differences in receptor usage among the many avian leukosis virus subgroups. We hypothesize that some retroviruses have altered receptor usage in response to selective pressures imposed by receptor polymorphisms in their hosts. To test this hypothesis, we passaged td-Pr-RSV-B on cocultured permissive chicken (C/E) and nonpermissive quail (QT6/BD) cells. A variant virus with an expanded host range was identified at passage 29 and ultimately shown to be identical in sequence to td-Pr-RSV-B, except for changes at codons 155 and 156 of SU amino acid corresponding to two amino acid changes within hr1. Superinfection resistance studies suggest that the variant virus recognizes the subgroup B receptor on chicken cells and the subgroup E receptor on quail cells. These findings indicate that altered receptor usage can be conferred by small changes in env and may point to a key region for receptor interaction. Further, they demonstrate the evolutionary potential of retroviral env genes to alter receptor usage in response to appropriate selective pressure.     

Resistance to infection by subgroups B,D, and E avian sarcoma and leukosis viruses is explained by a premature stop codon within a resistance allele of the tvb receptor gene

Here we present the first molecular characterization of the defect associated with an avian sarcoma and leukosis virus (ASLV) receptor resistance allele, tvb(r). We show that resistance to infection by subgroups B, D, and E ASLV is explained by the presence of a single base pair mutation that distinguishes this allele from tvb(s1), an allele which encodes a receptor for all three viral subgroups. This mutation generates an in-frame stop codon that is predicted to lead to the production of a severely truncated protein.     

Avian sarcoma and leukosis virus cytopathic effect in the absence of TVB death domain signaling

The cytopathic effect (CPE) seen with some subgroups of avian sarcoma and leukosis virus (ASLV) is associated with viral Env activation of the death-promoting activity of TVB (a tumor necrosis factor receptor-related receptor that is most closely related to mammalian TNF-related apoptosis-inducing ligand [TRAIL] receptors) and with viral superinfection leading to unintegrated viral DNA (UVD) accumulation, which is presumed to activate a cellular DNA damage response. In this study, we employed cells that express signaling-deficient ASLV receptors to demonstrate that an ASLV CPE can be uncoupled from the death-promoting functions of the TVB receptor. However, these cell-killing events were associated with much higher levels of viral superinfection and DNA accumulation than those seen when the virus used signaling-competent TVB receptors. These findings suggest that a putative cellular DNA damage response that is activated by UVD accumulation might act in concert with the death-signaling pathways activated by Env-TVB interactions to trigger cell death. Such a model is consistent with the well-established synergy that exists between TRAIL-signaling pathways and DNA damage responses which is currently being exploited in cancer therapy regimens.     

Low frequency of the Mx allele for viral resistance predates recent intensive selection in domestic chickens

Avian influenza is a serious threat to the poultry industry and, as the potential source of a human pandemic virus, to public health. Different Mx alleles have been reported to confer resistance or susceptibility to influenza virus replication, and so knowledge of their frequencies is important when considering the potential for improvement of modern commercial flocks. We analysed a range of chicken lines and ancestral breeds for the relevant Mx codon that confers resistance or susceptibility to influenza virus replication. We confirmed the high frequency of the susceptibility allele in contemporary meat-type (broiler) birds compared to egg-laying strains and found this difference is present already in ancestral breeds. We sequenced full-length complementary DNA (cDNA) and noted additional substitutions, which may be associated with the resistance haplotypes. High frequencies of the susceptibility allele could be readily reduced by modern breeding techniques.     

Adaptation of tobacco etch potyvirus to a susceptible ecotype of Arabidopsis thaliana capacitates it for systemic infection of resistant ecotypes

Viral pathogens continue to emerge among humans, domesticated animals and cultivated crops. The existence of genetic variance for resistance in the host population is crucial to the spread of an emerging virus. Models predict that rapid spread decreases with the frequency and diversity of resistance alleles in the host population. However, empirical tests of this hypothesis are scarce. Arabiodpsis thaliana—tobacco etch potyvirus (TEV) provides an experimentally suitable pathosystem to explore the interplay between genetic variation in host''s susceptibility and virus diversity. Systemic infection of A. thaliana with TEV is controlled by three dominant loci, with different ecotypes varying in susceptibility depending on the genetic constitution at these three loci. Here, we show that the TEV adaptation to a susceptible ecotype allowed the virus to successfully infect, replicate and induce symptoms in ecotypes that were fully resistant to the ancestral virus. The value of these results is twofold. First, we showed that the existence of partially susceptible individuals allows for the emerging virus to bypass resistance alleles that the virus has never encountered. Second, the concept of resistance genes may only be valid for a well-defined viral genotype but not for polymorphic viral populations.     

Low pH is required for avian sarcoma and leukosis virus Env-dependent viral penetration into the cytosol and not for viral uncoating

A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-beta-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating.     

Efficient subgroup C avian sarcoma and leukosis virus receptor activity requires the IgV domain of the Tvc receptor and proper display on the cell membrane

We recently identified and cloned the receptor for subgroup C avian sarcoma and leukosis viruses [ASLV(C)], i.e., Tvc, a protein most closely related to mammalian butyrophilins, which are members of the immunoglobulin protein family. The extracellular domain of Tvc contains two immunoglobulin-like domains, IgV and IgC, which presumably each contain a disulfide bond important for native function of the protein. In this study, we have begun to identify the functional determinants of Tvc responsible for ASLV(C) receptor activity. We found that the IgV domain of the Tvc receptor is responsible for interacting with the glycoprotein of ASLV(C). Additional experiments demonstrated that a domain was necessary as a spacer between the IgV domain and the membrane-spanning domain for efficient Tvc receptor activity, most likely to orient the IgV domain a proper distance from the cell membrane. The effects on ASLV(C) glycoprotein binding and infection efficiency were also studied by site-directed mutagenesis of the cysteine residues of Tvc as well as conserved amino acid residues of the IgV Tvc domain compared to other IgV domains. In this initial analysis of Tvc determinants important for interacting with ASLV(C) glycoproteins, at least two aromatic amino acid residues in the IgV domain of Tvc, Trp-48 and Tyr-105, were identified as critical for efficient ASLV(C) infection. Interestingly, one or more aromatic amino acid residues have been identified as critical determinants in the other ASLV(A-E) receptors for a proper interaction with ASLV glycoproteins. This suggests that the ASLV glycoproteins may share a common mechanism of receptor interaction with an aromatic residue(s) on the receptor critical for triggering conformational changes in SU that initiate the fusion process required for efficient virus infection.     

Mechanisms of avian retroviral host range extension

Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.     

The hr1 and fusion peptide regions of the subgroup B avian sarcoma and leukosis virus envelope glycoprotein influence low pH-dependent membrane fusion

The avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B) virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB) genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1) region of the surface (SU) EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM) EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation.     

Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.     

Genetics of post-hatching survival potential of Australorp chicks infected as embryos by subgroup A Rous sarcoma virus: further support to 4-allele genetic model.

Embryos (II day-old) of Australorp breed were inoculated via chorioallantoic membrane (CAM) with subgroup A Rous sarcoma virus, and hatched subsequently. The post-hatch survival period in chicks was recorded upto the last chick that died by virus-induced liver tumour, which had a range from 3 to 50 days with an average of 13 +/- 8.7 days. The survival potential of progency tested Australorp parents selected on the basis of negative CAM-infection and those selected on uninoculated embryos, differed significantly (P less than 0.01) while maintaining an inverse relationship between liver tumour mortality and degrees of infection of CAMs. The homozygous susceptibles lacking either ar1 or ar2 or both alleles of the tva (tumour virus a) locus died within 7 days of post-hatching, supporting thereby 4-allele genetic model of tva locus recently proposed for the control of LT- and CAM-infection phenotypes.     

HLA-A*01 allele: a risk factor for dengue haemorrhagic fever in Brazil's population

Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients.     

Induced liver tumour further support to a genetic marker with its high correlation with chorioallantoic membrane phenotypes in selected layer lines

The conventional chorioallantoic membrane (CAM) phenotype assay was conducted using 11-day-old embryonatic eggs of white Leghorn strains, each inoculated with 0.2 ml of subgroup A Rous sarcoma virus (RSV) and subgroup C RSV separately containing 10(3)pfu ml(-1). Eggs were further incubated for hatching. Harvesting of CAMs for counting of pocks and monitoring chicks for liver tumour (LT) mortality during 4 weeks of post-hatching period were followed. The conversely associated phenotype (CAP) incidence i.e. CAM(+) LT(-) and CAM(-) LT(+) was observed in all three lines for both subgroup A and C virus infection. The LT deaths of chicks in all strains occurred within 21 days post-hatch irrespective of virus subgroups. The regression analysis of %LT death (transformed data) distributed within pock count range (PCR) basis was performed. The regression coefficients (b(i)'s) were found to be non-significant, indicating that %LT death did not correlate with number of particles that entered the cells because the chicks that had at least 25 pock counts in CAMs died with few exceptions. This study upheld the view that the CAM phenotypes (S and R) under the control of a pair of alleles, a(s) and a(r) at the tva locus and c(s) and c(r) at the tvc locus as reported extensively. Because of a high correlation coefficients between CAM and LT phenotypes [S and LT(+)] in respect of subgroup A (r = 0.72) and subgroup C (r = 0.81) infection, it is obvious that one could postulate a pleiotropic control of the two traits by the tva and tvc genes, respectively. Indeed fitting a 4-allele model in both tva and tvc locus, suggesting that CAPs are the indicator for nullifying the conventional 2-allele model proposed for the induced tumour expression phenotypes by leukosis sarcoma viruses.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

HLA class I-driven evolution of human immunodeficiency virus type 1 subtype c proteome: immune escape and viral load

Human immunodeficiency virus type 1 (HIV-1) mutations that confer escape from cytotoxic T-lymphocyte (CTL) recognition can sometimes result in lower viral fitness. These mutations can then revert upon transmission to a new host in the absence of CTL-mediated immune selection pressure restricted by the HLA alleles of the prior host. To identify these potentially critical recognition points on the virus, we assessed HLA-driven viral evolution using three phylogenetic correction methods across full HIV-1 subtype C proteomes from a cohort of 261 South Africans and identified amino acids conferring either susceptibility or resistance to CTLs. A total of 558 CTL-susceptible and -resistant HLA-amino acid associations were identified and organized into 310 immunological sets (groups of individual associations related to a single HLA/epitope combination). Mutations away from seven susceptible residues, including four in Gag, were associated with lower plasma viral-RNA loads (q < 0.2 [where q is the expected false-discovery rate]) in individuals with the corresponding HLA alleles. The ratio of susceptible to resistant residues among those without the corresponding HLA alleles varied in the order Vpr > Gag > Rev > Pol > Nef > Vif > Tat > Env > Vpu (Fisher's exact test; P < or = 0.0009 for each comparison), suggesting the same ranking of fitness costs by genes associated with CTL escape. Significantly more HLA-B (chi(2); P = 3.59 x 10(-5)) and HLA-C (chi(2); P = 4.71 x 10(-6)) alleles were associated with amino acid changes than HLA-A, highlighting their importance in driving viral evolution. In conclusion, specific HIV-1 residues (enriched in Vpr, Gag, and Rev) and HLA alleles (particularly B and C) confer susceptibility to the CTL response and are likely to be important in the development of vaccines targeted to decrease the viral load.