Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160

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Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for

Restriction of Human Immunodeﬁciency Virus Type 1 (HIV-1):

HSV-1 gM Protein Potently Restricts HIV-1 by Preventing

Intracellular Transport and Processing of Env gp160

Sachith Polpitiya Arachchige,

a

Wyatt Henke,

a

Ankita Pramanik,

a

Maria Kalamvoki,

a

Edward B. Stephens

a

a_{Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center,} Kansas City, Kansas, USA

ABSTRACT

Virus-encoded proteins that impair or shut down speciﬁc host cell

func-tions during replication can be used as probes to identify potential

proteins/path-ways used in the replication of viruses from other families. We screened nine

pro-teins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict

human immunodeﬁciency virus type 1 (HIV-1) replication. We show that several

HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the

replica-tion of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we

ob-served that gM restriction of HIV-1 occurred through interference with the

process-ing and transport of gp160, resultprocess-ing in a signiﬁcantly reduced level of mature

gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking

the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160

processing nor the release of infectious virus. These studies identify proteins from

heterologous viruses that can restrict viruses through novel pathways.

IMPORTANCE

HIV-1 infection of humans results in AIDS, characterized by the loss of

CD4

⫹

T cells and increased susceptibility to opportunistic infections. Both HIV-1 and

HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus,

the identiﬁcation of HSV-1 proteins that directly restrict HIV-1 or interfere with

path-ways required for HIV-1 replication could lead to novel antiretroviral strategies. The

results of this study show that select viral proteins from HSV-1 can potently restrict

HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1

through a novel pathway by interfering with the processing of gp160 and its

incor-poration into virus maturing from the cell.

KEYWORDS

UL24, US3, gM, glycoprotein transport, herpes simplex virus, human

immunodeﬁciency virus, restriction

D

ominant-negative (DN) mutants have been used extensively to study the

impor-tance of viral proteins in replication and to decipher virus-host interactions. The

viral proteins from heterologous viruses that act in a dominant-negative fashion to

decrease the replication or release of infectious virus of another viral family may

provide alternative antiviral strategies. Herpes simplex virus 1 (HSV-1) is a major

opportunistic pathogen in human immunodeﬁciency virus type 1 (HIV-1)-infected

patients that may be a cofactor by interacting with HIV-1 at the cellular and/or

molecular level. HSV-1 is a highly cytopathic virus that is maintained in a latent state in

humans (1–3). The HSV-1 genome has more than 80 open reading frames (ORFs) that

encode proteins, including transactivators of transcription, DNA replication proteins,

RNA binding proteins, proteases, kinases, and structural components (viral

glycopro-teins and capsid proglycopro-teins). Due to the complex replication scheme of HSV-1, which

Received28 August 2017Accepted16

October 2017

Accepted manuscript posted online1

November 2017

CitationPolpitiya Arachchige S, Henke W,

Pramanik A, Kalamvoki M, Stephens EB. 2018. Analysis of select herpes simplex virus 1 (HSV-1) proteins for restriction of human immunodeﬁciency virus type 1 (HIV-1): HSV-1 gM protein potently restricts HIV-1 by preventing intracellular transport and processing of Env gp160. J Virol 92:e01476-17.

https://doi.org/10.1128/JVI.01476-17.

EditorRozanne M. Sandri-Goldin, University of

California, Irvine

crossm

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involves the cytoplasm, nucleus, and membrane organelles, HSV-1 encodes a unique

set of proteins that could act as dominant-negative proteins to block the replication or

release of a heterologous virus.

HIV-1 encodes proteins that facilitate replication and integration of the viral genome

into the host cell chromosome (i.e., reverse transcriptase and integrase) or viral

tran-scription and RNA export (i.e., Tat and Rev) (4) and that interact with host cell factors

that would normally restrict viral replication or promote immune responses against the

virus (i.e., Vpu, Nef, Vif, and Vpr) (5, 6). While many molecular aspects of HIV-1

replication have been deciphered, the identiﬁcation of novel cellular proteins/pathways

that affect HIV-1 replication continues to be explored.

While HIV-1 and HSV-1 generally do not infect the same target cells (HIV-1, CD4

⫹

T

cells and macrophages; HSV-1, epithelial cells and neurons), one exception is the central

nervous system (CNS). Both viruses are neurotropic, with HSV-1 the most common

cause of sporadic encephalitis in the United States and HIV-1 capable of causing overt

encephalitis and HIV-1-associated dementia (HAD) (7, 61). Despite current treatments

for HIV-1 (combined antiretroviral therapy [cART]), approximately 50% of infected

patients continue to develop HIV-1-associated neurocognitive disorders (HAND) (8–10).

Both HIV-1 and HSV-1 infect astrocytes and microglia of the CNS (11–18). Astrocytes are

the most abundant cells of the CNS and have several functions, including biochemical

support of brain endothelial cells essential to maintaining the blood-brain barrier,

production of neurotrophic immune-modulatory factors, neuronal homeostasis, and

repair and scarring following CNS trauma (19). Microglia account for 1 to 15% of all cells

found within the brain. As the resident macrophages, they function as the ﬁrst and

main form of active immune defense in the CNS (20). Thus, disruption of the function

of these types of cells and release of immune modulators toxic to neurons by viral

infection can have detrimental consequences for the CNS.

Here, we screened nine HSV-1 proteins for the ability to restrict the replication of

HIV-1. Our data show differential effects of these proteins on HIV-1 replication, with

three HSV-1 proteins, US3, UL24, and gM, potently restricting HIV-1 replication. We have

further characterized the novel mechanism through which HSV-1 gM restricts HIV-1

replication.

RESULTS

Identiﬁcation of HSV-1 proteins that potently restrict HIV-1.

We analyzed nine

HSV-1 proteins for the ability to restrict HIV-1. These proteins were ICP0, VP16, VP22,

US3, US11, gM, UL24, UL46, and UL47. The proteins have different functions in the

HSV-1 life cycle, which are listed in Table 1. We ﬁrst analyzed the expression of these

proteins to ensure that they were expressed well in 293 cells. Transfection of 293 cells

with each plasmid construct and analysis of expression at 48 h posttransfection using

anti-myc, anti-Flag, or protein-speciﬁc antibodies and Western blotting showed that all

nine proteins were expressed (Fig. 1). We then constructed a pcDNA3.1(

⫹

) vector

expressing a human codon-optimized glycoprotein M (gM) with a hemagglutinin (HA)

tag at the N terminus that was expressed well in 293 cells for further experiments (Fig.

1E). We next cotransfected 293 cells with each of the above-mentioned vectors and a

vector with the complete genome of HIV-1 strain NL4-3 (pNL4-3). At 48 h

posttrans-fection, the levels of infectious virus and p24 released into the culture medium were

determined. The results indicated that three proteins, VP16, UL47, and US11, had no

signiﬁcant effect on HIV-1 replication and release of infectious virus (Fig. 2A). Three

other HSV-1 proteins, ICP0, UL46, and VP22, resulted in an intermediate level of

restriction (

⬃

7 to 14% that of the control) (Fig. 2A). Finally, three HSV-1 proteins, US3,

UL24, and gM, restricted the release of infectious virus with averages of 0.08, 0.45, and

0.04% of the pcDNA3.1(

⫹

) control restriction, respectively (Fig. 2A). HA-gM and myc-gM

restricted HIV-1 at equivalent levels. Viability assays using the Zombie Aqua ﬁxable

assay kit (BioLegend) revealed that over 98% of the cells transfected with HA-gM,

myc-UL24, and myc-US3 were viable at 48 h (data not shown). The levels of p24

released from cultures generally correlated with levels of infectious virus released. US3

Polpitiya Arachchige et al. Journal of Virology

January 2018 Volume 92 Issue 2 e01476-17 jvi.asm.org 2

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and UL24 released p24 at levels that were 0.75 and 0.15%, respectively, of the pcDNA3.1

control level (Fig. 2B). In contrast, the levels of p24 released from cells transfected with

the vectors expressing HA-gM and pNL4-3 were 46.3% of the control level (Fig. 2B).

These discordant results suggested that HSV-1 gM restriction of HIV-1 might be due to

interaction with a speciﬁc HIV-1 protein. We next analyzed the expression of HIV-1

proteins in the presence of the individual HSV-1 proteins. 293 cells were cotransfected

as described above and at 24 h starved for methionine/cysteine and radiolabeled for 6

h. Cell lysates were prepared, and aliquots were used to immunoprecipitate HIV-1

proteins,

␤

-actin, and individual HSV-1 proteins (Fig. 2C to E). Companion cultures were

cotransfected with pcDNA3.1(

⫹

) and with vectors expressing each HSV-1 protein for

comparative purposes (Fig. 2F). Our results indicated that, with the exceptions of gM,

US3, and UL24, the synthesis of HIV-1 proteins was similar to that of the pNL4-3/

pcDNA3.1(

⫹

) control. Additionally, expression of the HSV-1 proteins in the presence of

pNL4-3 was not signiﬁcantly different from that of companion cultures expressing only

the HSV-1 protein, indicating that the HIV-1 proteins did not result in degradation of

TABLE 1HSV-1 gene products, classes of transcripts, functions, and locations in HSV-1-infected cells

Gene Protein Transcript class Function Location HIV-1 restrictiona

RL2 ICP0 Alpha (␣) Promiscuous transactivator; disrupts DNA repressor complexes; E3 ligase; blocks innate immunity

Nucleus, cytoplasm ⫹

UL10 gM Gamma (␥2) Efﬁcient viral entry; regulates capsid envelopment; redirects surface proteins totrans-Golgi

Nucleolus,trans-Golgi ⫹⫹⫹

UL24 UL24 Gamma (␥1) Nucleolar dispersal of host proteins; nuclear egress; polykaryocyte formation in the absence of UL24

Nucleus; membrane association

⫹⫹⫹

UL46 VP11/12 Gamma (␥1) Tegument; modulates VP16 transactivation function; PI3Kb_/AKT

activation; blocks innate immunity through STING

Nucleus, cytoplasm ⫹⫹

UL47 VP13/14 Gamma (␥1) Tegument; RNA binding protein; modulates the activity of Vhs; modulates VP16 transactivation function

Shuttles between nucleus and cytoplasm

⫺

UL48 VP16 Gamma (␥) Tegument; induces␣genes; virion assembly; together with VP22 blocks Vhs; interacts with gH

Nucleus, cytoplasm ⫺

UL49 VP22 Gamma (␥) Tegument; with VP16 neutralizes Vhs; binds RNA; chromatin remodeling

Nucleus, cytoplasm ⫹

US3 US3 Beta (␤) Tegument; serine/threonine protein kinase (PKA-like) Nucleus, cytoplasm ⫹⫹⫹ US11 US11 Gamma (␥2) Tegument; RNA binding protein; antagonizes PKR; inhibits

RIG-I and MDA-5; capsid egress

Nucleolus, cytoplasm ⫺

a_{Level of HIV-1 restriction:}_⫺_{, 0 to 30% restriction;}_⫹_{, 90 to 31% restriction;}_⫹⫹_{, 91 to 99% restriction;}_⫹⫹⫹_,_⬎_{99% restriction.} b_{PI3K, phosphatidylinositol 3-kinase.}

FIG 1Expression of HSV proteins in 293 cells. 293 cells were transfected with vectors expressing different HSV-1 proteins or the empty vector. At 48 h, cell lysates were prepared and processed for Western blotting. Proteins were detected using antibodies to either myc- or Flag-tagged HSV-1 proteins. (A) Western blot showing expression of myc-tagged HSV-1 proteins. (B) Western blot showing expression of Flag-tagged HSV-1 proteins. (C) Western blot showing expression of ICP0. (D) Western blot showing expression of VP16. (E) Western blot showing expression of myc-gM and HA-gM. The gels at the bottom show the loading controls (␤-actin) for the Western blots.

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FIG 2Identiﬁcation of HSV-1 proteins that potently restricted HIV-1. 293 cells were cotransfected with either the empty-vector plasmid or plasmids expressing each HSV-1 protein (ICP0, US3, US11, myc-gM, HA-gM, UL24, UL46, UL47, VP16, or VP22) and a plasmid with the HIV-1 genome (pNL4-3). At 48 h, the culture supernatants were collected. (A and B) The levels of infectious virus released into the culture supernatants were determined using TZM.bl cell assays, and p24 levels in the culture supernatants were determined using p24 antigen capture assays. (A) Levels of infectious virus released into the culture medium from cells cotransfected with the pNL4-3 genome and plasmids expressing the different HSV-1 proteins. (B) Levels of p24 protein produced

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the HSV-1 proteins tested. Taken together, these results indicate that ectopic

expres-sion of select HSV-1 proteins potently restricted the production of infectious HIV-1.

Processing and release of viral proteins in the presence of US3, UL24, and gM.

As US3, UL24, and gM restricted the production of infectious HIV-1 to the greatest

extent, we examined the processing and release of the HIV-1 proteins in cells

express-ing these HSV-1 proteins. 293 cells were cotransfected with either the empty

pcDNA3.1(

⫹

) vector or one expressing HA-US3, HA-UL24, HA-gM, or UL47 and with

pNL4-3. The transfected cells were starved for 2 h and radiolabeled with [

35

S]methio-nine/cysteine. The viral proteins immunoprecipitated from the culture medium and

from cell lysates at 16 h postlabeling. Cells cotransfected with pcDNA3.1(

⫹

) and pNL4-3

expressed gp160, gp120, p55 Gag, and p24 in the cell lysates, while gp120 and p24

were readily detected in the supernatants from culture medium (Fig. 3A to D). In cells

transfected with HA-US3 or HA-UL24 and pNL4-3, the detection of viral proteins by

immunoprecipitation was signiﬁcantly decreased, with only minor amounts of p55

detected in the cell lysates and no viral proteins detected in the supernatants from

culture medium (Fig. 3A and B). However, in cells cotransfected with HA-gM and

pNL4-3, p24 was readily detectable in cell lysates and supernatants from culture

medium, although at reduced levels compared to the pcDNA3.1(

⫹

)/pNL4-3 control

(Fig. 3C). Additionally, in HA-gM/pNL4-3 cultures, while the gp160 precursor was

detected in cell lysates, no gp120 was observed in cell lysates, which was also reﬂected

in the levels of gp120 released from cells (Fig. 3C). Finally, in myc-UL47/NL4-3 cultures,

p24 and gp120 were released into the culture medium at levels similar to those of the

pcDNA3.1(

⫹

)/pNL4-3 control (Fig. 3D). These results correlated well with the p24

assays.

Inoculation of HA-gM-expressing cultures with infectious virus restricts HIV-1,

but not VSV.

To determine if gM would restrict infectious HIV-1, we generated

vesicular stomatitis virus G protein (VSV-G)-pseudotyped virus. 293 cells were

trans-fected with either the empty pcDNA3.1(

⫹

) vector or one expressing HA-gM. The cells

were then inoculated with VSV-G/NL4-3 (multiplicity of infection [MOI]

⫽

0.01), and the

levels of infectious virus released were determined using TZM.bl cells (Fig. 4A) while

those of p24 were determined by antigen capture assays. The results indicated that

the levels of infectious virus released were signiﬁcantly decreased (9.9% those of the

empty-vector control). We also examined whether VSV replication was restricted in the

presence of HA-gM. 293 cells were transfected with either the empty vector or one

expressing HA-gM, and at 24 h posttransfection, the cells were inoculated with VSV. At

24 h postinoculation, the virus-containing culture medium was collected and titrated

on 293 cells. Our results showed that the presence of HA-gM did not restrict VSV

replication, indicating the speciﬁcity of the HA-gM-mediated HIV-1 restriction (Fig. 4C).

gM from HSV-2 also restricts HIV-1.

We also analyzed the ability of the gM protein

from HSV-2 to restrict HIV-1. We chose HSV-2 gM for sequence similarity to HSV-1

(

⬃

80% at the amino acid level). Both proteins were expressed well in 293, as

deter-mined by Western blotting (Fig. 5A). The slight decrease in relative molecular weight

(

M

r

) of myc-tagged gM of HSV-2 (myc-gM[HSV-2]) was due to a 7-amino-acid deletion

FIG 2Legend (Continued)

from cells cotransfected with the pNL4-3 genome and plasmids expressing the different HSV-1 proteins. The experiments were performed at least four times, and statistical differences with the pcDNA3.1(⫹)/HIV-1 control were evaluated using a two-tailed Studentttest, with aPvalue of⬍0.05 (Œ) considered signiﬁcant. The error bars indicate standard deviations and the numbers the mean levels of infectious virus released compared to the pcDNA3.1(⫹) control. (C to E) Expression of HIV-1 proteins in the presence of HSV-1 proteins. 293 cells were transfected with pcDNA3.1(⫹) or a vector expressing each HSV-1 protein and a plasmid expressing the pNL4-3 viral genome. At 24 h, the cells were starved and radiolabeled with [35_{S]methionine/cysteine for 6 h, at} which time the culture medium was removed and cell lysates were prepared. Equal aliquots of the cell lysates were immuno-precipitated with antibodies against the HIV-1 proteins (C),␤-actin (D), or HSV-1 proteins (E). (F) Companion cultures from the experiments in panel C to E were cotransfected with the empty vector pcDNA3.1(⫹) and vectors expressing each of the HSV-1 proteins. 293 cells were cotransfected with pcDNA3.1(⫹) and vectors expressing each HSV-1 protein listed above. At 24 h, the cells were starved and radiolabeled with [35_{S]methionine/cysteine for 6 h. The culture medium was removed, and cell lysates were} prepared. Equal aliquots of the cell lysates were immunoprecipitated with appropriate antibodies against the HSV-1 proteins. The numbers to the left indicate the position of the molecular weight markers (in thousands).

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(amino acids 384 to 390) in the C-terminal domain. While the levels of p24 were similar

in cells transfected with HA-gM[HSV-1], myc-gM[HSV-2], and pNL4-3, we observed that

myc-gM[HSV-2] was also capable of signiﬁcant restriction of HIV-1 replication at levels

similar to those of HA-gM[HSV-1] (Fig. 5B and C).

Pulse-chase analyses reveal that HIV-1 gp160 is inefﬁciently processed and

degraded in the presence of HSV-1 gM.

As the above-described results suggested

that gp160 was inefﬁciently processed, we determined if expression of HA-gM would

prevent the processing of gp160 in the presence or absence of other viral proteins. In

the ﬁrst set of experiments, 293 cells were cotransfected with the empty pcDNA3.1(

⫹

)

vector or the vector expressing HA-gM and plasmid pNL4-3. At 48 h posttransfection,

the cells were starved for methionine/cysteine and radiolabeled, and pulse-chase

experiments were performed with chase periods of 0, 1, 3, and 6 h (Fig. 6A and B). The

culture medium was harvested, and cell lysates were prepared as described in Materials

and Methods. HIV-1 proteins were immunoprecipitated using an anti-simian-human

immunodeﬁciency virus (SHIV) serum and an antibody directed against p24. As shown

FIG 3Processing and release of HIV-1 proteins in the presence of US3, UL24, and gM. 293 cells were cotransfected with the empty pcDNA3.1(⫹) vector or one expressing myc-UL24, myc-US3, HA-gM, or myc-UL47 and a plasmid containing the HIV-1 NL4-3 genome (pNL4-3). At 24 h, the cells were incubated in medium lacking methionine/cysteine for 2 h, followed by radiolabeling the cultures with [35_{S]methionine/cysteine for 16 h. The culture medium was harvested, cell lysates were} prepared as described in Materials and Methods, and HIV-1 Env and Gag proteins were immunoprecipitated with appropriate antibodies. In addition, HSV-1 proteins (Us3, myc-UL24, and UL47) immunoprecipitated with anti-myc and gM immunopre-cipitated with anti-HA are shown below panels A to D. The immunoprecipitates were collected on protein A-Sepharose, washed, and boiled in sample reducing buffer. The proteins were separated on 10% SDS gels and visualized using standard radiographic techniques. (A) myc-US3. (B) Myc-UL24. (C) HA-gM. (D) myc-UL47. Numbers to the left indicate the position of molecular weight markers (in thousands).

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in Fig. 6A, in the presence of the empty vector, gp160 and p55 were detected in the 0-h

chase period, with gp120 and p24 detected at the 1-, 3-, and 6-h chase periods in the

cell lysates. Both gp120 and p24 were detected in the cell culture medium at 1-, 3-, and

6-h chase periods. Pulse-chase analysis of cells transfected with HA-gM and pNL4-3

resulted in gp160 and p55 being detected in the 0-h chase period and little to no gp120

being immunoprecipitated from cell lysates in the 3- and 6-h chase periods (Fig. 6B).

This was also reﬂected in the HIV-1 proteins immunoprecipitated from the culture

medium. Almost no gp120 was detected in the culture, although p24 was clearly

released into the culture medium (Fig. 6B).

In a second pulse-chase experiment, 293 cells were cotransfected with a vector

expressing HA-gM and the pBal.26 vector. Pulse-chase analyses were performed

simi-FIG 4HSV-1 gM restricts the production of infectious HIV-1 but not VSV. (A) Levels of infectious HIV-1 produced from cells transfected with the empty vector pcDNA3.1(⫹) or one expressing HA-gM and inoculated with VSV-G/NL4-3. 293 cells were transfected with the empty vector pcDNA3.1(⫹) or one expressing HA-gM. At 24 h posttransfection, the cells were inoculated with VSV-G/NL4-3 at an MOI of 0.01. At 72 h posttransfection, the cell culture medium was collected, clariﬁed by low-speed centrifugation, and analyzed for infectious virus using TZM.bl cell assays and for p24 levels using p24 antigen capture assays. (B) Levels of p24 released from transfected/infected cells from panel A. (C) Levels of infectious VSV released into culture supernatants from cells transfected with the empty vector pcDNA3.1(⫹) or one expressing HA-gM and inoculated with VSV. 293 cells were transfected as for panel A, and at 24 h posttransfection were inoculated with VSV (MOI⫽0.01). The virus was collected 24 h after virus inoculation, clariﬁed, and titrated for levels of infectious virus. The results shown represent the means from two experiments. The error bars indicate standard deviations.

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larly to those with the virus-transfected cells discussed above, except with chase

periods of 0, 1, 3, and 6 h (Fig. 7). HIV-1 Env was immunoprecipitated using anti-Env

antibodies. Our results indicated that in cells cotransfected with the empty vector

pcDNA3.1(

⫹

) and the vector expressing HIV-1 strain Bal Env, gp160 was synthesized

and processed into gp120 by the 1-h chase period in cell lysates and was barely

detected in the culture medium at the 1-h chase period but was detected at later time

points (Fig. 7A). In contrast, the processing of gp160 into gp120 was not detected in

cells cotransfected with vectors expressing HA-gM and HIV-1 Bal Env glycoprotein until

the 6-h chase period, and the levels of gp120 were reduced in the culture medium in

the 6-h chase period (Fig. 7B).

Intracellular localization of Env in the presence of HSV-1 HA-gM protein.

The

intracellular expression of HIV-1 Env was analyzed in the absence or presence of gM. We

ﬁrst examined the expression of gM in the absence of HIV-1 proteins. COS-7 cells were

transfected with the empty pcDNA3.1(

⫹

) vector or the vector expressing HA-gM and

examined by immunoﬂuorescence for its intracellular localization. At 24 h, the cells

were ﬁxed, permeabilized, and stained with an antibody against the HA tag and

antibodies against calnexin (endoplasmic reticulum [ER]), Golgin 97 (Golgi apparatus),

or TGN46 (

trans

-Golgi network [TGN]). Our results indicated that gM partially

colocal-ized with calnexin, Golgin 97, and TGN46, as was previously reported (Fig. 8A to I). We

did not observe signiﬁcant amounts of HA-gM on the cell plasma membrane. We next

analyzed the cells cotransfected with either the pcDNA3.1(

⫹

) vector or one expressing

HA-gM and a vector expressing the Bal Env (pBal.26). Our results indicated that after

cotransfection of the vector expressing HA-gM with pBal.26, the level of Env expression

was signiﬁcantly diminished, with most of the gp160 staining occurring in intracellular

compartments (Fig. 9A to C). In contrast, cotransfection of cells with the empty

pcDNA3.1(

⫹

) vector and the pBal.26 vector resulted in Env that was easily detected

in intracellular compartments and on the cell plasma membrane (Fig. 9G to I). These

results agree with the pulse-chase analysis showing decreased expression with

time.

FIG 5The gM from HSV-2 also restricts HIV-1 replication. 293 cells were cotransfected with the pcDNA3.1(⫹)vector or one expressing HA-gM[HSV-1], myc-gM[HSV-2], or HSV-1 myc-UL47 and a plasmid containing the NL4-3 genome. At 24 h posttransfection with pNL4-3, the culture medium was collected and assayed for release of p24 and for infectious virus using TZM.bl assays. The experiments were performed at least three times, and statistical differences with the pcDNA3.1(⫹)/HIV-1 control were evaluated using a two-tailed Studentttest, with aPvalue of⬍0.05 considered signiﬁcant. (A) Expression of HA-gM[HSV-1] and myc-gM[HSV-2] proteins in 293 cells. (B) Levels of p24 protein produced from cells cotransfected with pcDNA3.1(⫹), HA-gM[HSV-1], or myc-gM[HSV-2] and a plasmid with the pNL4-3 genome. (C) Levels of infectious virus in the culture medium from cells cotransfected with pcDNA3.1(⫹), HA-gM[HSV-1], or myc-gM[HSV-2] and a plasmid with the pNL4-3 genome. The error bars indicate standard deviations.

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Truncation of gM abrogates restriction of HIV-1.

A current model of the structure

of HSV-1 gM suggests that the protein is a type III membrane protein that spans the

membrane eight times, with both the N and C termini located on the cytoplasmic side

of the membrane (11, 21–24). We determined if removal of the C-terminal domain

would abrogate gM restriction of HIV-1. A mutant was constructed in which the

C-terminal 126 amino acids were deleted from gM (just after the last transmembrane

domain). This mutant, HA-gM[Δ345-473], was expressed well in 293 cells (Fig. 10A).

When the culture medium was analyzed for p24, we observed that the levels of p24 in

the medium of cells transfected with HA-gM[Δ345-473] were similar to those with the

empty-vector control (Fig. 10B). When used in restriction assays with pNL4-3, our results

indicated that, unlike the full-length protein, HA-gM[Δ345-473] was unable to restrict

HIV-1 (Fig. 10C). We also determined if the processing of Env was normal in the absence

FIG 6HSV-1 gM inhibits the processing of HIV-1 gp160 and release of gp120. 293 cells were cotransfected with the empty pcDNA3.1(⫹) vector or one expressing HA-gM and pNL4-3. At 48 h posttransfection, the cells were starved for methionine/cysteine and radiolabeled with [35_{S]methionine/cysteine for 30 min. The} radiolabel was removed, and the cells were washed twice and chased in medium containing 100⫻ methionine/cysteine for either 0, 1, 3, or 6 h. The culture medium was removed, and cell lysates were prepared as described in Materials and Methods. HIV-1 Env and Gag proteins were immunoprecipitated with appropriate antibodies, and the immunoprecipitates were collected on protein A-Sepharose. Samples were washed and then boiled in sample reducing buffer. The proteins were separated on 10% SDS gels and visualized using standard radiographic techniques. (A) HIV-1 proteins immunoprecipitated from cell lysates and culture medium from 293 cells cotransfected with pcDNA3.1(⫹) and pNL4-3. (B) HIV-1 proteins immunoprecipitated from cell lysates and culture medium from 293 cells cotransfected with vectors expressing HA-gM and pNL4-3. Numbers in the middle of the panels indicate the position of the molecular weight markers (in thousands).

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of other viral proteins and HA-gM[Δ345-473]. 293 cells were cotransfected with either

the empty pcDNA3.1(

⫹

) vector or one expressing HA-gM or HA-gM[Δ345-473] and with

vector pNL4-3. At 48 h, the cells were starved and radiolabeled with [

35

S]methionine/

cysteine for 30 min, and the radiolabel was chased in cold excess methionine/cysteine

for 6 h. HIV-1 proteins were immunoprecipitated with anti-SHIV and anti-p24

antibod-FIG 7HSV-1 gM inhibits the processing of HIV-1 gp160 and release of gp120 in the absence of other HIV-1 proteins. 293 cells were cotransfected with the empty pcDNA3.1(⫹) vector or one expressing HA-gM and pBalEnv26, which expresses the Bal strain Env gp160 protein. At 48 h posttransfection, the cells were incubated in medium lacking methionine/cysteine for 2 h, followed by radiolabeling the cultures with [35_{S]methionine/cysteine for 30 min. The radiolabel was removed, and the cells were} washed twice and chased in medium containing 100⫻methionine/cysteine for either 0, 1, 3, or 6 h. The culture medium was removed, and cell lysates were prepared as described in Materials and Methods. HIV-1 Env proteins were immunoprecipitated with an anti-gp160 antibody, and the immunoprecipitates were collected on protein A-Sepharose. Samples were washed and boiled in sample reducing buffer, and proteins were separated on 10% SDS gels and visualized using standard radiographic techniques. (A) HIV-1 Env proteins immunoprecipitated from cell lysates and culture medium from 293 cells cotrans-fected with pcDNA3.1(⫹) and pBalEnv. (B) HIV-1 Env proteins immunoprecipitated from cell lysates and culture medium from 293 cells cotransfected with vectors expressing HA-gM and pBalEnv.

FIG 8Intracellular localization of HA-gM. COS-7 cells on coverslips were transfected with the vector expressing HA-gM. At 24 h posttransfection, the cells were fixed, permeabilized, and blocked as described in Materials and Methods. The cells were reacted with antibodies against calnexin (ER), Golgin 97 (Golgi complex), or TGN46 (trans-Golgi network); washed; and reacted with secondary antibodies as described in Materials and Methods. The cells were then reacted with a rabbit anti-HA-FITC antibody to identify the gM proteins. The cells were washed and counterstained with DAPI for 5 min. All the coverslips were mounted and examined with a Leica TCS SPE confocal microscope with a 63⫻objective and a 2⫻digital zoom using the LAS X software package. A 488-nm filter was used to visualize FITC, a 647-nm filter to visualize Alexa Fluor 647, and a 405-nm filter to visualize DAPI staining. (A,D, and G) Cells stained for HA-gM. (B, E, and H) Cells stained for calnexin (B), Golgin 97 (E), and TGN46 (H). (C, F, and I) Merge of panels A and B (C), of panels D and E (F), and of panels G and H (I). The bar in panel A represents 10␮m.

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ies. The results of the Env immunoprecipitation from cell lysates revealed that gp120

was released from cells cotransfected with pcDNA3.1(

⫹

) and pNL4-3 (Fig. 10D and E).

In contrast, cells cotransfected with vectors expressing HA-gM and pNL4-3 showed very

little gp120 released into the culture medium. Finally, cotransfection of cells with

HA-gM[Δ345-473] and pNL4-3 revealed release of gp120 into the culture medium at

levels similar to those with the empty vector (Fig. 10D and E). We also examined cells

cotransfected with the vector expressing HA-gM[Δ345-473] and the pBal.26 vector by

immunoﬂuorescence (Fig. 9D to F). Our results indicated that expression of Env was

very similar to that observed with cells cotransfected with the empty vector,

pcDNA3.1(

⫹

), and pBal.26 (Fig. 9G to I). Taken together, these results indicate that the

C-terminal domain of gM is required to restrict Env transport and for release of

infectious HIV-1 from cells.

DISCUSSION

Previous studies have shown that herpesviruses can reactivate HIV-1, although this

phenomenon depended on the herpesvirus, experimental model, and cell line used (2,

25–31). Several HSV-1 proteins encoded by immediate-early (IE) genes, such as ICP0

(infected cell polypeptide 0), ICP4, and ICP27, have been shown to transactivate the

HIV-1 long terminal repeat (LTR) (26, 29, 30, 32, 33). More recently, it was shown that

CEM cells chronically infected with either HIV-1 (CEM

HIV

) or HSV-1 (CEM

HSV

) had

different effects on the replication of HIV-1 or HSV-1 (34). The investigators showed that

superinfection of CEM

HIV

cells with HSV-1 resulted in an increase in the release of virus

from the cells as measured by reverse transcriptase activity. This effect was inversely

proportional to the multiplicity of infection used to inoculate cultures. In contrast, HIV-1

superinfection of CEM

HSV

cells resulted in a delay in the kinetics of HIV-1 replication,

suggesting a negative inﬂuence of HSV-1 on HIV-1 replication (34). To date, no studies

FIG 9Intracellular expression of HIV-1 gp160 in the presence of HA-gM or HA-gM[Δ345-473]. COS-7 cells were cotransfected with either the pcDNA3.1(⫹) vector, one expressing HA-gM, or one expressing HA-gM[Δ345-473] and a plasmid expressing the gp160 (pBal.26). At 24 h posttransfection, the cells were fixed, permeabilized, and blocked as described in Materials and Methods. The cells were then reacted with a goat anti-gp160 antibody to identify the gp160 proteins and with anti-HA to identify gM, washed, and reacted with secondary antibody as described in the text. The coverslips were counterstained with DAPI for 5 min. All the coverslips were mounted and examined with a Leica TCS SPE confocal microscope with a 63⫻objective and a 2⫻digital zoom using the LAS X software package. A 488-nm filter was used to visualize FITC, a 647-nm filter to visualize Alexa Fluor 647, and a 405-nm filter to visualize DAPI staining. (A to C) Cells cotransfected with vectors expressing HA-gM and Bal Env. (D to F) Cells cotransfected with vectors expressing HA-gM[Δ345-473] and Bal Env. (G to I) Cells cotransfected with vector pcDNA3.1(⫹) and Bal Env. (A, D, and G) Cells stained for HA-containing proteins. (B, E, and H) Cells stained for gp160. (C, F, and I) Merge of panels A and B (C), of panels D and E (F), and of panels G and

H (I). The bar in panel A represents 10␮m.

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FIG 10Truncation of gM abrogates restriction of HIV-1 replication. 293 cells were cotransfected with either the empty pcDNA3.1(⫹) vector or one expressing full-length HA-gM or HA-gM[Δ345-473] and with pNL4-3. At 48 h, the culture supernatants were collected and cell lysates were prepared. The levels of p24 determined in the culture supernatants were quantiﬁed using p24 antigen capture assays, and equivalent levels of p24 were used to determine virus infectivity using TZM.bl cell assays. (A) Immunoprecipitation analysis showing the expression of HSV-1 HA-gM and HA-gM[Δ345-473]. 293 cells were transfected with pcDNA3.1(⫹) expressing HA-gM[Δ345-473] (lane 1), empty pcDNA3.1(⫹) (lane 2), or HA-gM (lane 3). At 24 h posttransfection, cells were starved for methionine/ cysteine and radiolabeled with [35_{S]methionine/cysteine for 6 h. Cell lysates were prepared, and} immunoprecipitations were performed with an anti-HA polyclonal serum. (B) Levels of p24 protein produced from cells cotransfected with pcDNA3.1(⫹), HA-gM, or HA-gM[Δ345-473] and the pNL4-3 genome. (C) Levels of infectious virus released into the culture medium from cells cotransfected with pcDNA3.1(⫹), HA-gM, or HA-gM[Δ345-473] and pNL4-3. (D and E) Pulse-chase analysis of 293 cells cotransfected with the empty pcDNA3.1(⫹) vector and pNL4-3, a vector expressing HA-gM and pNL4-3, a vector expressing HA-gM[Δ345-473] and pNL4-3. At 48 h posttransfection, the cells were starved for methionine/cysteine and radiolabeled with [35_{S]methionine/cysteine. The radiolabel was} chased for 0 or 6 h, and viral proteins immunoprecipitated from the cell lysates (D) or cell culture medium (E) as described in Materials and Methods. The error bars indicate standard deviations. Numbers to the left indicate the positions of the molecular weight markers (in thousands).

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have critically examined whether HSV-1 proteins could speciﬁcally restrict the

replica-tion of HIV-1. We explored the hypothesis that proteins expressed by HSV-1 could

impair the replication of HIV-1. We screened nine HSV-1 proteins by transfection for the

ability to restrict the replication of HIV-1. Of these proteins, US3 and UL24 profoundly

restricted HIV-1 replication, as determined by infectivity, viral protein biosynthesis, and

p24 release from cells.

In addition, our results indicated that HSV-1 and HSV-2 gM proteins restricted HIV-1

replication. In this study, we further analyzed the mechanism by which the HSV-1 gM

protein restricted HIV-1. Thirteen envelope glycoproteins that are involved in different

aspects of the viral replication cycle, including viral entry, egress from the nucleus, and

acquisition of the ﬁnal envelope, are known to be incorporated into HSV-1 virions.

HSV-1 gM is a 473-amino-acid type III transmembrane protein that synthesizes as a

47-kDa precursor that matures into a family of 53- to 63-kDa glycosylated proteins (35,

36). Ectopic expression of HSV-1 gM results in the protein being targeted to the TGN

(37). While a detailed topological map of gM is lacking, the available evidence predicts

that it has eight membrane-spanning domains, with the N- and C-terminal ends

oriented toward the cytosol (22, 37, 38). In a recent study, the roles of several gM

domains were analyzed with respect to the subcellular distribution (23). The

investi-gators found that expression of a gM protein consisting of amino acids 19 to 343

resulted in localization to the TGN, while the expression of other truncated proteins,

such as gM 1 to 342, lacking the ﬁnal residue of the last transmembrane domain, and

gM 133 to 473, lacking the ﬁrst two transmembrane domains, resulted in the retention

of gM in the ER (23). These data suggested that all the transmembrane helices and the

C-terminal domain were required for transport to the TGN (23). While gM is not

essential for HSV-1 replication in cell culture, its deletion reduces viral yields by

approximately 10-fold (35, 39). HSV gM is known to interact with multiple viral proteins,

including gN (24, 40, 41). HSV-1 gN is a 91-amino-acid ER-resident protein when

expressed in the absence of other HSV proteins (24). However, in the presence of gM,

gN is transported to the TGN and requires the presence of the hydrophobic core (i.e.,

the region with eight transmembrane helices) (24). Site-directed mutagenesis showed

that gM and gN interact through the formation of a disulﬁde bond between C-46 of gN

and C-59 of gM, although the disulﬁde bond is not necessary for transport to the TGN

(24). The gM glycoprotein also interacts with other HSV-1 glycoproteins, such as gB, gD,

and gH/gL. The interaction of these glycoproteins results in redirection to the TGN, the

site of ﬁnal envelopment (37). Additionally, gM can interact with and cause moderate

antagonism of BST-2 (tetherin), leading to enhanced HSV-1 release from cells (42, 43).

BST-2 is a protein that is antagonized by HIV-1 Vpu to facilitate virus release from cells

(44, 45). In another study, it was shown that BST-2 restricted the release of infectious

HSV-2 particles into the extracellular medium and that the HSV-2 glycoproteins gB, gD,

gH, and gL downregulated the endogenous expression of BST-2. Interestingly, HSV-2

gM did not downregulate the expression of BST-2 (46).

A salient question we have attempted to address in this study is what the

mecha-nism by which gM restricts HIV-1 is. Our results indicate that gp160 was not efﬁciently

cleaved into gp120/gp41, which occurs in the Golgi complex (47, 48). gp160 appeared

to be degraded with time, and very low levels of gp120 were observed in the culture

medium (and presumably the virus). One potential explanation for these observations

is that gM may perturb the transport of membrane proteins from the ER to the Golgi

complex. While we cannot totally rule out this scenario, other HSV-1 proteins are

synthesized and transported to the cell surface in the presence of gM. Also, the

production of infectious VSV from cells transfected with the empty pcDNA3.1(

⫹

) vector

or one expressing HA-gM was not affected, suggesting that the presence of gM had no

impact on the well-studied VSV G protein. Another potential explanation is that gM

may interact with gp160 within the rough endoplasmic reticulum (RER) to prevent

transport of gp160 to the Golgi complex and subsequent processing into gp120/gp41.

The HSV-1 gM contains four tyrosine-based motifs with the consensus sequence YXX

⌽

(where X is any amino acid and

⌽

is a bulky hydrophobic amino acid) and an acidic

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cluster (DEXXDDXXD). YXX

⌽

motifs, either alone or in combination with acidic clusters,

play crucial roles in efﬁcient transport of the well-studied VSV-G protein and of the gB

proteins of different herpesviruses from the ER to the Golgi complex (21, 49–52).

Consistent with this hypothesis, the expression and processing of gp160 in the

pres-ence of the HA-gM[Δ345-473] mutant, lacking the C-terminal 129 amino acids, was

similar to transfection with the empty vector, indicating that the C-terminal domain of

gM is important for the inhibition of Env transport and processing. Alternatively, if gM

interacts with gp160, it could prevent proper folding of gp160. This in turn could signal

an endoplasmic reticulum-associated protein degradation response, leading to the

degradation of gp160 (53, 54). A working model is that the cytoplasmic domain of

HA-gM may interact with the cytoplasmic domain of gp160 to mask the tyrosine-based

motifs and or acidic clusters of gM that are essential for transport to the TGN. A

consequence of this interaction would be decreased transport of gp160 to the Golgi

complex and subsequent processing. This would decrease the levels of gp120/gp41

transported to the cell plasma membrane and incorporated into virions, resulting in the

restriction of HIV-1 spread through a novel mechanism. These studies also indicate that

the proteins from heterologous viruses, such as the herpesviruses, may provide

infor-mation on unique mechanisms of restriction that could possibly be used in the

development of novel antiviral approaches.

MATERIALS AND METHODS

Cells, plasmids, and viruses.293 cells were used for transfection of vectors expressing the HSV-1 proteins. The TZM-bl cell line, which was used as an indicator to measure virus infectivity, was obtained from the NIH AIDS Reagent Branch (55–58). A plasmid expressing Rev and gp160 from HIV-1 Env (HIV-1 strain Bal; pBal.26) and a plasmid with the entire HIV-1 NL4-3 genome (pNL4-3) were obtained from the NIH AIDS Reagent Branch. Both cell lines were maintained in Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum (R10FBS), 10 mM HEPES buffer, pH 7.3, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 5␮g gentamicin. The HSV-1 proteins analyzed in this study were expressed as either untagged proteins (ICP0 and VP16), with a myc tag (US3, gM, UL24, UL46, UL47, and VP22), or with a Flag tag (US11) fused to the N termini of the proteins. All HSV-1 ORFs were PCR amplified from the HSV-1 (F) genome, and the sequences were verified by sequence analysis. A vector [pcDNA3.1(⫹)] expressing human codon-optimized HA-tagged HSV-1 gM was synthesized by GenScript, and myc-tagged HSV-2 gM was obtained from Origene (catalog number VC100874). An HSV-1 gM mutant in which the C-terminal 128 amino acids were deleted (HA-gM[Δ345-473]) was constructed by GenScript and expressed in pcDNA3.1(⫹). The sequence was confirmed by sequence analysis. Expression of HSV-1 proteins was confirmed by transfection with the TurboFect transfection reagent (ThermoFisher) and Western blot analysis using a mouse monoclonal antibody directed against the Flag tag (Sigma; F3165), a mouse monoclonal antibody against the c-myc tag (Santa Cruz; 9E10; sc-40), or rabbit antibodies against the individual proteins (ICP0 and VP16).

Analysis of virus production in the presence of HSV-1 proteins.To analyze the virus restriction properties of the HSV-1 proteins, 293 cells were transfected with either empty pcDNA3.1(⫹) vector or a vector expressing one of the HSV-1 proteins and with pNL4-3. At 48 h posttransfection, the culture medium was collected and clarified by low-speed centrifugation, and the supernatant was analyzed for p24 using antigen capture assays. The levels of infectious virus in the culture supernatants were titrated on TZM.bl cells (59, 60). TZM.bl cells have an integrated copy of a retrovirus vector expressing ␤-galactosidase under the control of the HIV-1 LTR. If TZM.bl cells are infected with HIV-1, Tat protein expression will result in the expression of␤-galactosidase, and infected cells can be visualized by fixing and staining with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside). All assays were performed at least four times and analyzed for statistical significance using a two-tiered Studentttest with cells cotransfected with the empty vector pcDNA3.1(⫹) and pNL4-3 set at 100% infectivity.

Biosynthesis and processing of viral proteins in the presence of HSV-1 proteins.The biosyn-thesis and processing of viral proteins were examined in the presence of US3, UL24, and gM. 293 cells were cotransfected with empty pcDNA3.1(⫹) or pcDNA3.1(⫹) expressing HA-US3, HA-UL24, or HA-gM and with pNL4-3. At 24 h, the cells were washed and incubated in DMEM without methionine/cysteine for 2 h. The cells were washed and radiolabeled in DMEM containing 500␮Ci [35_{S]Translabel (methionine} and cysteine; MP Biomedical) for 16 h. The culture medium was collected, subjected to low-speed centrifugation, and made 1⫻with respect to radioimmunoprecipitation assay (RIPA) buffer (1⫻RIPA buffer is 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5% deoxycholate, 0.2% SDS, 10 mM EDTA). Cell lysates were prepared by lysis in RIPA buffer for 10 min on ice. The lysates were centrifuged in a microcentrifuge at 14,000 rpm for 10 min, and the supernatants were transferred to fresh tubes. HIV-1 proteins were immunoprecipitated using a monkey anti-SHIV antibody (which recognizes the gp160/gp120 and gp41 proteins of HIV-1) and a monoclonal antibody against p24 (number 24-4 from the NIH AIDS Reagent Program). The immunoprecipitates were collected by incubation with protein A-Sepharose beads at 4°C. The beads were washed three times with RIPA buffer, and the samples were resuspended in sample

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reducing buffer. The samples were boiled, and the proteins were separated by SDS-PAGE (10% gel) and visualized using standard radiographic techniques.

Restriction of infectious-virus production by HA-gM.Following the initial screen, we examined HSV-1 gM for the ability to restrict the replication of infectious virus. We generated infectious HIV-1 NL4-3 pseudotyped with the VSV-G protein by cotransfecting 293 cells with a vector expressing VSV-G protein and one containing the full-length HIV-1 NL4-3 genome. At 48 h, the culture medium was collected, clariﬁed by low-speed centrifugation (1,200 rpm; 10 min), and frozen at⫺86°C. The number of infectious units was determined using 293 cells, serial 10-fold dilutions of virus, and p24 assays. To determine if HA-gM would restrict the production of infectious NL4-3, 293 cells were transfected with the vector expressing HA-gM. At 24 h posttransfection, the cultures were inoculated with VSV-G/NL4-3 at a multiplicity of infection of 0.01. At 72 h posttransfection, the levels of infectious virus in the culture supernatants were titrated on TZM.bl cells as described above (59, 60). The 50% tissue culture infectious doses (TCID50) of virus in TZM.bl cells were determined, and the numbers of TCID50in the empty vector and the vector expressing HA-gM were compared. All assays were performed at least four times and analyzed for statistical signiﬁcance using two-tiered Studentttests.

To examine the speciﬁcity of HA-gM restriction, we also analyzed the ability of HA-gM to restrict VSV. 293 cells were transfected with the empty vector or one expressing HA-gM. At 24 h posttransfection, the cells were inoculated with VSV (MOI⫽0.01) for 1 h, at which time the inoculum was removed and the cells were washed three times. The infected cells were incubated in medium for 24 h, at which time the culture medium was removed and centrifuged at 1,200 rpm for 10 min. The levels of infectious virus in the culture supernatants were titrated using 10-fold dilutions and 293 cells in 96-well plates. The TCID50of virus in the 293 cells was determined, and the numbers of TCID50in the empty vector and the vector expressing HA-gM were compared.

Pulse-chase analysis of biosynthesis and processing of viral proteins in the presence of HSV-1 gM.293 cells were transfected with the empty pcDNA3.1(⫹) vector or pcDNA3.1(⫹) expressing HA-gM and pNL4-3. At 48 h, the cells were washed and incubated in medium without methionine/cysteine for 2 h, followed by radiolabeling with 500␮Ci [35_{S]methionine/cysteine (Translabel) for 30 min. The medium} was removed, and the cells were washed twice in medium containing 100_⫻excess cold methionine and cysteine and then incubated in the same medium for 0, 1, 3, and 6 h. At various times, the culture medium was collected, subjected to low-speed centrifugation, and made 1⫻with respect to RIPA. The cell lysates were prepared as described above. HIV-1 proteins were immunoprecipitated and processed, and analysis was performed as described above. In addition, the synthesis, processing, and transport of HIV-1 Env were also analyzed in the absence of other HIV-1 proteins. 293 cells were cotransfected with plasmid pBal.26, which expresses HIV-1 Env (strain Bal), and either empty pcDNA3.1(⫹) vector or one expressing HA-gM. The pulse-chase analysis was performed as described above, except that goat anti-gp160 antibodies were used in the immunoprecipitation assays (NIH AIDS Reagent Program; catalog numbers 189 and 51).

Intracellular localization of HIV-1 Env in the presence of HSV-1 gM. We determined if the intracellular localization of HIV-1 Env was altered in the presence of HSV-1 gM. We first determined the intracellular localization of gM protein in the absence of HIV-1. COS-7 cells were plated on 13-mm coverslips in 6-well plates and transiently transfected with the empty pcDNA3.1(⫹) vector or one expressing HA-gM protein using TurboFect transfection reagent (ThermoFisher) according to the man-ufacturer’s instructions. The cultures were maintained for 24 h, and the medium was removed; the cultures were washed three times in PBS (pH 7.4) and fixed in 4% paraformaldehyde (prepared in PBS) for 30 min. The coverslips were washed in PBS, permeabilized in 0.1% Triton X-100, washed, and incubated in PBS containing 5% BSA for 1 h. For colocalization of gM and the endoplasmic reticulum, the coverslips were reacted with a mouse monoclonal antibody against calnexin (mouse anti-calnexin; Novus Biologicals; 3H4A7) overnight at 4°C. The coverslips were washed and reacted with a secondary rabbit anti-mouse IgG Alexa Fluor 594 (Abcam ab150128). The coverslips were then reacted with rabbit anti-HA-fluorescein isothiocyanate (FITC) (Abcam; ab1208) and then counterstained with DAPI (4= ,6-diamidino-2-phenylindole) (1␮g/ml) for 5 min. For colocalization of HA-gM with the Golgi complex or TGN, the coverslips were reacted with rabbit anti-Golgin 97 (Abcam; ab84340) or rabbit anti-TGN46 (Novus Biologicals; NBP1-49643). The coverslips were washed and reacted with goat anti-rabbit IgG Alexa Fluor 647 (Abcam; ab150083), washed, and reacted with rabbit anti-HA-FITC (Abcam; ab1208) for 1 h. The coverslips were counterstained with DAPI (1␮g/ml) for 5 min. All the coverslips were washed and mounted in glycerol-containing mounting medium (SlowFade antifade solution A; Invitrogen). The coverslips were examined using a Leica TCS SPE confocal microscope with a 63⫻objective and a 2⫻ digital zoom using the Leica Application Suite X (LAS X) software package. A 405-nm filter was used to visualize DAPI staining, a 488-nm filter was used to visualize FITC, and a 647-nm filter was used to visualize Alexa Fluor 647. A minimum of 50 cells were examined for each sample, and the results shown in the figures are representative of each sample.

To analyze the intracellular localization of gp160 in the presence or absence of gM proteins, COS-7 cells were cotransfected with either empty pcDNA3.1(⫹) vector or one expressing HA-gM or HA-gM[Δ345-473] and pBal.26Env. At 24 h, the cells on coverslips were washed, ﬁxed, permeabilized, and blocked as described above. The coverslips were reacted with a mixture of three goat anti-gp160 antibodies (NIH AIDS Reagents Branch; catalog numbers 51, 189, and 191) and with mouse monoclonal anti-HA antibody (SC7392) overnight at 4°C. The coverslips were washed and then reacted with donkey anti-goat IgG Alex Fluor 568 antibody (Abcam; ab175704) and Alexa Fluor 488 rabbit anti-mouse IgG (Thermo Fisher; A11059) for 2 h and then counterstained with DAPI (1␮g/ml) for 5 min. The coverslips

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were mounted, and a minimum of 50 cells were examined using the appropriate ﬁlters as described above.

ACKNOWLEDGMENTS

This work was supported by NIH grant R21 AI108391 to E.B.S.

We thank the Northwestern Sequence facility for sequence analyses and the KUMC

Biotechnology Core for oligonucleotide synthesis. The HIV-1 p24 Gag monoclonal

antibody (number 24-4) from Michael H. Malim was obtained through the NIH AIDS

Reagent Program, Division of AIDS, NIAID, NIH. pNL4-3 was obtained from Malcolm

Martin through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. The HIV-1

BaL.26 clone was obtained from J. R. Mascola through the NIH AIDS Reagent Program,

Division of AIDS, NIAID (catalog number 11446).

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