Definition of VPU sensitivity using a model VPU target and role of hydrophobicity of the membrane spanning domain in the viral envelope glycoprotein fusogenicity

Retroviral compatibility with diverse glycoproteins has been known and identified through the course of several studies. However, molecular mechanisms of glycoprotein acquisition are poorly defined. Glycoproteins are acquired by the virus as it buds out of the cell at the plasma membrane. Budding of retroviruses involves multiple interactions between viral and cellular proteins and a mature viral particle is the consummation of a regulated and a sequential process. Currently there are no drugs to target the assembly step of retrovirus. In the series of studies outlined here, we outline a physical factor, Vpu that contributes to glycoprotein exclusion from HIV particles. Using a model Vpu target, Gibbon ape Leukemia Virus (GaLV) Env, we have deduced the characteristics of a protein that is targeted by Vpu through its cytoplasmic tail domain (CTD). This unique observation of Vpu modulating the GaLV Env CTD allowed us to compare the two modes of Vpu mediated protein modulation- CTD mediated and membrane spanning domain (MSD) mediated. Subsequently, we studied the contribution of MSD hydrophobicity to Env recruitment to viral budding sites. Curiously, although hydrophobicity of MSD did not dictate Env recruitment, the helicity changes as a result of our mutations resulted in observation of the Env fusogenicity

Functional complementation of a model target to study Vpu sensitivity.

HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the 'Vpu sensitivity sequence' in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex

Mixed Env trimers can be Vpu sensitive.

<p>Sensitivity to Vpu of each complementation pair is indicated. The degree of Vpu sensitivity is indicated within parentheses as a ratio of infectivity in the absence of Vpu to infectivity in the presence of Vpu. Data shown is the average of three independent experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068507#pone-0068507-g002" target="_blank">Fig 2B</a>.Vpu^R- Vpu resistant; Vpu^S- Vpu sensitive.</p

Defective Env pairs with different CTDs complement each other functionally.

<p>(A) Infectivity of the indicated Envs relative to infectivity with F-MLV Env. Data shown is the average of two experiments. (B) Infectivity of complementation pairs in the presence or absence of Vpu, relative to infectivity with F-MLV Env. (C) Average of three experiments with the infectivity normalized to that of F-MLV Env. Error bars indicate SD in the experiments. Welch's T- test was performed to determine p- values, indicative of statistical significance. NS- Non significant; BD- Binding defective; FD- Fusion defective.</p

Schematic of the gammaretroviral Env proteins.

<p>The amino acid sequences in the CTD of F-MLV Env and the F-MLV/GaLV Env chimera are depicted. Truncations in the CTD of the Envs, F-MLV Δ25 and F-MLV/GaLV Δ8 are indicated. Underlined sequence represents the element that dictates Vpu sensitivity. The cartoon has been adapted from figure 1 of ref. 5.</p

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo

The Antiviral Factor SERINC5 Impairs the Expression of Non-Self-DNA

SERINC5 is a restriction factor that becomes incorporated into nascent retroviral particles, impairing their ability to infect target cells. In turn, retroviruses have evolved countermeasures against SERINC5. For instance, the primate lentiviruses (HIV and SIV) use Nef, Moloney Murine Leukemia Virus (MLV) uses GlycoGag, and Equine Infectious Anemia Virus (EIAV) uses S2 to remove SERINC5 from the plasma membrane, preventing its incorporation into progeny virions. Recent studies have shown that SERINC5 also restricts other viruses, such as Hepatitis B Virus (HBV) and Classical Swine Fever Virus (CSFV), although through a different mechanism, suggesting that SERINC5 can interfere with multiple stages of the virus life cycle. To investigate whether SERINC5 can also impact other steps of the replication cycle of HIV, the effects of SERINC5 on viral transcripts, proteins, and virus progeny size were studied. Here, we report that SERINC5 causes significant defects in HIV gene expression, which impacts virion production. While the underlying mechanism is still unknown, we found that the restriction occurs at the transcriptional level and similarly affects plasmid and non-integrated proviral DNA (ectopic or non-self-DNA). However, SERINC5 causes no defects in the expression of viral RNA, host genes, or proviral DNA that is integrated in the cellular genome. Hence, our findings reveal that SERINC5’s actions in host defense extend beyond blocking virus entry

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δnef-Infected Animals

ABSTRACT Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239Δnef. Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef. In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef. IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin